Heat shock protein 27 is involved in PCV2 infection in PK-15 cells

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Heat shock protein 27 is involved in PCV2 infection in PK-15 cells

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Jie Liu, Lili Zhang, Xuejiao Zhu, Juan Bai, Liming Wang, Xianwei Wang, Ping Jiang ∗ Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

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Article history: Received 16 January 2014 Received in revised form 25 May 2014 Accepted 27 May 2014 Available online xxx

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Keywords: Porcine circovirus type 2 PK-15 cells Hsp27 Phosphorylated forms

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1. Introduction

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Porcine circovirus type 2 (PCV2) has been identified as the etiologic agent which causing postweaning multisystemic wasting syndrome in swine farms in the world. Some quantitative proteomic studies showed that many proteins significantly changed in PCV2-infected cells. To explore the role of cellular chaperones during PCV2 infection, cytoprotective chaperone Hsp27 was analyzed in PCV2-infected PK-15 cells in this study. The results showed that Hsp27 could up-regulate and accumulate in phosphorylated forms in the nuclear zone during PCV2 replication. Suppression of Hsp27 phosphorylation with specific chemical inhibitors or downregulation of all forms of Hsp27 via RNA interference significantly reduced the virus replication. Meanwhile, over-expression of Hsp27 enhanced PCV2 genome replication and virion production. It indicated that Hsp27 was required for PCV2 production in PK-15 cells culture. It should be helpful for understanding the mechanism of replication and pathogenesis of PCV2 and development of novel antiviral therapies in the future. © 2014 Published by Elsevier B.V.

Porcine circovirus type 2 (PCV2) is also an immunosuppressive virus in pigs. It is a small, nonenveloped, single-stranded DNA virus that belongs to the circoviridae family (Tischer et al., 1982). The virus contains two major open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes the replication proteins which are involved in virus replication, and ORF2 encodes the capsid (Cap) protein (Liu et al., 2001; Nawagitgul et al., 2000). PCV2 has been identified as the etiologic agent of the postweaning multisystemic wasting syndrome (PMWS) (Allan et al., 1998; Ellis et al., 1998), that is widely spread in swine farms and represents one of several porcine circovirus associated diseases (PCVAD). PCV2 infection usually accompanies lymphocyte or monocyte depletion and thus further results in immune suppression in the disease (Lee et al., 2010; Shibahara et al., 2000). The immunosuppressive disease mainly presents as PMWS, which caused a great economic loss worldwide (Bolin et al., 2001; Krakowka et al., 2000). However, the immunosuppressive and pathogenic mechanisms have remained unclear completely in PCV2-infected pigs. Due to the small genome size and the limited coding capacity, it is generally acknowledged that the viral life cycle of PCV2

∗ Corresponding author. Tel.: +86 25 84395504; fax: +86 25 84396640. E-mail addresses: [email protected], [email protected] (P. Jiang).

must depend extensively on host factors. For elucidation of the interaction between host cells and PCV2, proteome analysis had been utilized for host cellular responses to virus infection. Zhang et al. (Zhang et al., 2009) identified 34 host-encoded proteins that were altered in PCV2-infected PK-15 cells using two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF, while Fan and colleagues (Fan et al., 2012) detected 163 proteins that were significantly affected in PCV2-infected PK-15 cells with the SILAC-based approach. In our previous study, we described quantitative proteomic analysis of a highly permissive PK-15 cell line (produced by our laboratory) infected with PCV2 using isobaric tags for relative and absolute quantification (iTRAQ), combined with multidimensional liquid chromatography (LC) and tandem MS analysis, and found many heat shock proteins, including Hsp90, Hsp70, Hsp60 and Hsp27 (Data not shown). Previously, we firstly found the positive effect of heat stress on the replication of PCV2 in the continuous porcine monocytic cellline 3D4/31. Hsp70 played an important role in that process. Meanwhile, Hsp27 also significantly changed during PCV2 infection (Liu et al., 2013b). Thus, we suspected that Hsp27 might play a certain role in the process of PCV2 infection. In this study, it was firstly found that Hsp27 was up-regulated in PCV2-infected highly permissive PK-15 cells. Down-regulation of Hsp27 via RNA interference and up-regulation of Hsp27 by transfection of a recombinant plasmid expressed Hsp27 indicated that Hsp27 play important role in PCV2 replication the PK-15 cells.

http://dx.doi.org/10.1016/j.virusres.2014.05.024 0168-1702/© 2014 Published by Elsevier B.V.

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2. Materials and methods

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2.1. Cell culture and virus infection

mounted onto microscope slides, and samples examined under a Zeiss LSM700 confocal microscope. 2.4. siRNA experiments

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Porcine circovirus type 2 strain, WG09 (GenBank accession no.: GQ845027), was isolated from an intensive pig farm in Shanghai, China, in 2009. The virus stock was a twenty-fourth-passage cell culture prepared in PK-15 cells clone with a titer of 106.5 TCID50 /mL. PK-15 cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (GIBCO, Invitrogen Corporation, CA). Cells were seeded in 25 cm2 culture flasks (Costar, Corning Incorporated, NY) until 75% confluence. Next, cells were inoculated with PCV2 WG09 strain at 1 MOI and collected at 12, 24, 48 and 96 hours post-inoculation (hpi), respectively. The amount of fetal bovine serum in medium was decreased to 2%. Uninfected cells served as the mock infection group. Viral propagation was confirmed via Western Blot using a monoclonal antibody against PCV2 Cap protein (made in our laboratory). The p38 inhibitor, SB203580 (SB), was purchased from Sigma Chemical (St. Louis, MO) and prepared according to the manufacturer’s instructions. SB was used at a concentration of 30 ␮M.

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2.2. Western blot

2.5. Recombinant plasmid-mediated Hsp27 expression Recombinant eukaryotic expression plasmid (pcDNA3.1Hsp27-Flag) encoding Flag-tagged Hsp27 (GenBank accession No. NM 001007518) was constructed by our lab. PK-15 cells were seeded in 24 well plates before 24 h transfection and transfected with the Flag-tagged plasmids or empty plasmid (pcDNA3.1) by lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cells were infected with PCV2 at MOI of 1, and new medium with 1% FBS was added. After 24 hpi, Western blotting, quantitative RT-PCR assays and TCID50 assays were used to detect the effect of over-expressed Hsp27 on PCV2 replication.

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Samples of PCV2-infected and uninfected PK-15 cells were lysed at 12, 24, 48, and 96 hpi, and the protein concentrations determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Product No. 23227, USA). Equivalent amounts of cell lysate proteins were subjected to 12% SDS-PAGE and transferred to 0.22 ␮m nitrocellulose membranes (Hybond-C extra, Amersham Biosciences). After blotting, membranes were incubated at 37 ◦ C for 60 min, respectively, with mouse monoclonal antibodies (mAbs) to actin (Abcam, Cambridge, UK), Hsp90 (Abcam, Cambridge, UK), Hsp27 (Abcam, Cambridge, UK), PCV2 Cap protein (made in our laboratory) or rabbit polyclonal antibody to Hsp27-phosphor S15 (Abcam, Cambridge, UK), Hsp27-phosphor S78 (Abcam, Cambridge, UK), and Hsp27-phosphor S82 (Abcam, Cambridge, UK). After washing three times with 0.05% PBST, membranes were incubated at 37 ◦ C for 60 min with horseradish peroxidase-conjugated goat anti-mouse IgG (Boshide, Wuhan, China) or goat anti-rabbit IgG (Boshide, Wuhan, China). Detection was performed using chemiluminescence luminol reagents (SuperSignalWest PicoTrial Kit, Pierce). Western blot analyses were repeated by three times. Protein spot levels were determined using ImageJ quantification software. The level of relative proteins were quantified by immunoblot scanning and normalized with respect to the amount of ␤-actin (lower panel).

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2.3. Confocal microscopy

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Infected or mock-infected PK-15 cells were cultured on glass coverslips. Cells were processed for indirect immunofluorescence at the indicated time-points by fixing in 4% paraformaldehyde for 30 min. Cell membranes were permeabilized by incubating in PBS buffer containing 0.2% Triton X-100 for 5 min. Samples were blocked in 1% BSA for 30 min before incubation with primary and secondary antibodies. All samples were washed using PBS. PCV2 Cap protein was detected with mouse mAb anti-Cap (generated in our laboratory) at 1:200 dilution. Hsp27 was detected with rabbit polyclonal anti-Hsp27 (Santa Cruz, CA). The phosphorylation state of Hsp27 was detected with the rabbit polyclonal antibody antiHsp27-phosphor S15, Hsp27-phosphor S78 and Hsp27-phosphor S82 at 1:200 dilutions. The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-mouse (Beyotime) and Alexa Fluor 555-conjugated goat anti-rabbit (Beyotime). Coverslips were

All siRNAs (Invitrogen) were transfected using Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer’s instructions. PK-15 cells (in serum-free DMEM) were transfected with 200 pmol/well of siRNA-Hsp27 or nonspecific siRNA. After 6 h at 37 ◦ C, medium supplemented with 2% FCS was added. Cells were incubated for 24 h, washed with Hank’s medium, and infected with PCV2 at 1 MOI. At 48 h post-infection, cell samples were harvested. Levels of Hsp27 protein and viral production after siRNA transfection were analyzed via immunoblotting, quantitative RT-PCR assays and TCID50 . The siRNA sequences were as follows: siRNA-1Hsp27 (S1), 5 -GGAUGAGCACGGCUUCAUU-3 ; siRNA-2 Hsp27 (S2), 5 -GCUUCAUUUCCCGGUGUUU-3 ; siRNA-3 Hsp27 (S3), 5 -UCACCAUCCCUGUCACUUU-3 ; nonspecific control (NC), 5 -UUCUCCGAACGUGUCACGU-3 (scrambled S1 oligonucleotides).

2.6. Quantitative RT-PCR assays Total DNA from cultured cells was isolated using the TaKaRa DNA Mini kit (TaKaRa, China) following the manufacturer’s instructions, and assayed via real-time PCR, as described earlier (Feng et al., 2008). The sense primer PCV2F (5 -CCAGGAGGGCGTTCTGACT-3 ), antisense primer PCV2R (5 -CGTTACCGCTGGAGAAGGAA-3 ) and probe (5 -FAM-AATGGCATCTTCAACACCCGCCTC-TARAM-3 ) were used to amplify PCV2 DNA. Quantitative real-time PCR was carried out using ABI7300 v.1.3 (ABI). Total RNA was extracted from cellular samples using TRIzol reagent (Invitrogen). Reverse transcription was carried out using M-MLV Reverse Transcriptase (Promega) using the manufacturer’s instructions, as described previously (Li et al., 2009). The RT reaction mixture (2 ␮l) was subjected to quantitative RT-PCR (Q-PCR) using Hsp27-specific primers (sense: 5 -GGAGATCACGGGCAAGCA3 ; antisense: 5 -GTGAAACACCGGGAAATGAAG-3 ), or ␤-actin (sense: 5 -TCTTCCAGCCCTCCTTCCT-3 ; antisense: 5 ACGTCGCACTTCATGATCGA-3 ) and SYBR Green Real-time PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan), according to the manufacturer’s recommendations. The reaction conditions were as follows: 95 ◦ C for 2 min, followed by 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 60 s. Cycle times of the internal reference that varied by >1.0 unit in triplicate were discarded. The relative amount of target gene mRNA was normalized to that of ␤-actin mRNA in the same sample. To confirm specific amplification, melting curve analysis of RT-PCR products was performed according to the manufacturer’s protocol. Q-PCR was performed in an ABI PRISM 7300 sequence detection system and analyzed with ABI PRISM 7300 SDS software (Applied Biosystems).

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Fig. 1. Investigation the changes of Hsp90, Hsp27 and viral Cap protein in PCV2-infected PK-15 using Western blot analysis. The levels of relative proteins were quantified with immunoblot scanning and normalized to the amount of ␤-actin (lower panel).

Q3 Fig. 2. Hsp27 localization in PCV2-infected cells. PK-15 cells grown on coverslips were infected with PCV2 (MOI of 1) and fixed at 12, 24, 48 and 96 hpi before immunofluorescence. Staining profiles for DNA using DAPI (blue), the host chaperone Hsp27 (red) and Cap protein (green), are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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2.7. Virus titration via immunofluorescence assay (IFA)

2.8. Statistical analysis

The PCV2 sample titer was determined via IFA in 96-well plates. Briefly, 100 ␮l cell suspensions were inoculated into semiconfluent monolayers of PK-15 cells and examined 72 h later for infection using IFA using standard protocols (McNair et al., 2004).

Statistical analysis was performed using GraphPad PRIM software (version 5.02 for Windows; GraphPad software Inc.). Data were analyzed to establish their significance using one-way or twoway analysis of variance (ANOVA), followed by the least-significant

Fig. 3. Western blotting analysis of phosphorylated Hsp27 in PCV2-infected or mock-infected cell lysates. Samples at 0, 4, 8, 12, 24, 48 and 96 hpi time-points were collected. The results at 0 hpi were the same as those in mock groups. The level of relative proteins was quantified using immunoblot scanning and normalized to the amount of ␤-actin (lower panel).

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Fig. 4. Immunofluorescence analysis of phospho-reactive species of Hsp27 in PCV2-infected cells. Immunofluorescence analysis of cellular chaperones at various time-points in uninfected or PCV2-infected cells (A–C) is shown. Infected cells were collected at 12, 24, 48 and 96 h after infection with PCV2. Uninfected cells were fixed at 96 h after the start of infection. Staining profiles for phospho-reactive Hsp27 species (red), viral Cap protein (green), and DNA using DAPI (blue) are shown. Merged images are shown in the last column. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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difference test, and expressed as means ± SD. Differences were regarded as significant at P < 0.01 (**).

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3. Results

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3.1. Hsp27 and Hsp90 changes in protein levels via Western blot

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To evaluate the differentially expression of Hsp27 and Hsp90 after PCV2 infection, the equal amounts of cell lysate protein

from PCV2-infected PK-15 and mock cells were tested by Western blot with the antibodies against Hsp27, Hsp90 and Cap protein, respectively. As shown in Fig. 1, the relative levels of Hsp27 in infected cells at 48 and 96 h post infection (hpi) were 1.5-fold higher than those at 12 and 24 hpi (P < 0.05). Meanwhile, the relative levels of Hsp90 in PCV2-infected PK-15 at 96 hpi were 2fold higher than those early time points (P < 0.05). It showed that Hsp27 and Hsp90 were up-regulation in PK-15 cells during PCV2 replication.

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Fig. 5. Western blotting analysis of the effect of the chemical inhibitor on phosphorylated Hsp27 in PCV2-infected or mock-infected cell lysates. Cells were left untreated (-SB) or treated with SB. Samples at 4, 8, 12, 24, 48 and 96 hpi were collected. The relative protein levels were quantified via immunoblot scanning and normalized to the amount of ␤-actin (lower panel).

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3.2. Subcellular localization of Hsp27 in PCV2-infected cells Since Hsp27 plays several roles that are potentially beneficial to the virus and coordinates the transfer of misfolded proteins to other chaperone systems, we further determined its subcellular distribution in PCV2-infected cells. Localization of Hsp27 in PK15 cells was analyzed via confocal microscopy using a specific rabbit polyclonal antibody. Hsp27 distribution showed dynamic changes during the course of PCV2 infection (Fig. 2). In uninfected cells, Hsp27 was detected as a diffuse staining pattern throughout cells. In infected cells, marked changes in the pattern were observed at advanced stages of infection at 24–96 hpi. At these time-points, Hsp27 showed accumulation in the nuclear zone, where is the viral replication compartments. It demonstrated that Hsp27 accumulated in the nucleus where viral replication occurs, but there was no obvious co-localization between Hsp27 and Cap protein.

3.4. Phosphorylated Hsp27 is accumulated in the nuclei of PCV2-infected cells Next, we examined subcellular localization of the phosphorylated forms of Hsp27 in infected cells. In uninfected cells, strong cytoplasmic staining for Hsp27-p-Ser15, Hsp27-p-Ser78 and Hsp27-p-Ser82 forms was detected (Mock; Fig. 4A–C). In PCV2-infected cells, strong nuclear staining for Hsp27-p-Ser15 and Hsp27-p-Ser78 forms was observed between 24 and 96 hpi (Fig. 4A and B). However, Hsp27 phosphorylated at Ser82 mainly remained in the cytoplasm of infected cells (Fig. 4C). The results indicated that Hsp27-p-Ser15 and Hsp27-p-Ser78 translocated into the nucleus at 24 hpi and accumulated in the nuclear zone during viral Cap protein expressed between 24 and 96 hpi. These findings were consistent with the confocal microscopy data on subcellular localization of Hsp27. 3.5. The phosphorylation state of Hsp27 affects PCV2 replication

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3.3. Hsp27 is phosphorylated during PCV2 infection The various functions of human Hsp27 are performed by different oligomeric species, and the balance among these species governed by mitogen-activated stress kinases within the cell. During stress conditions, the stress-activated kinase, p38, phosphorylates Hsp27 at any of three serines (Ser15, Ser78, and/or Ser82), which triggers dissociation from the higher-order oligomer (Rogalla et al., 1999). The phosphorylated forms perform auxiliary roles within stressed cells. Here, we analyzed the phosphorylation state of Hsp27 during PCV2 infection using polyclonal antibodies specific for each of the phosphorylated forms. Experiments with phospho-specific Hsp27 antibodies revealed that Hsp27 is phosphorylated at Ser15 and Ser78 between 4 and 96 hpi. Western blot analysis indicated no dramatic changes in the overall levels of phosphorylation at Ser82 (Fig. 3).

Further, we aimed to define whether the phosphorylation state of Hsp27 affects PCV2 replication. Cells were treated with SB, a chemical inhibitor of p38, the kinase that phosphorylates Hsp27 during periods of stress. Considering the influence of the half-life of SB, the medium was refreshed with or without SB every 24 h. As shown in Fig. 5, compared to the control groups treated without SB, Hsp27 phosphorylation was blocked in the groups treated with the p38 inhibitor. Furthermore, the viral Cap protein level was reduced, indicating that the inhibition affects PCV2 replication. Protein spot levels were determined using ImageJ quantification software. 3.6. RNAi-mediated depletion of Hsp27 reduces PCV2 viral yields To address the role of Hsp27 during PCV2 infection, we employed an RNAi approach to deplete the Hsp27 level and determined the consequences on viral yield. PK-15 cells were transfected

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Fig. 6. Hsp27 knockdown and PCV2 virus yield in Hsp27-depleted PK-15 cells. (A) Western blot analysis of Hsp27 protein in cells treated with either Hsp27 siRNA (S1–S3) or nonspecific siRNA (nc). Untreated infected (UT) cells are shown. The level of relative proteins was quantified via immunoblot scanning and normalized to the amount of ␤-actin (lower panel). (B) Western blot analysis of Hsp27 and Cap proteins in PCV2-infected cells treated with Hsp27 siRNA (S1–S3) or nonspecific siRNA (nc). Untreated infected (UT) cells are shown. The relative protein levels were quantified via immunoblot scanning and normalized to the amount of ␤-actin (lower panel). (C) Hsp27 mRNA and PCV2 Cap gene were detected using quantitative RT-PCR. Hsp27 mRNA was normalized to that of ␤-actin in the same sample. (D) Cell cultures were harvested and PCV2 titers detected as TCID50 .

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with siRNAs (S1, S2 and S3), either specific for the Hsp27 sequence or nonspecific (NC), and the Hsp27 level monitored via Western blotting and real-time PCR detection. S1, S2 and S3 had specific inhibitory effects (Fig. 6A). PK-15 cells were transfected with 200 pmol Hsp27 siRNA for 24 h, and cells infected with PCV2 at 1 MOI. In infected cells transfected with Hsp27 siRNA, Hsp27 was efficiently downregulated, compared to those transfected with control and scrambled siRNA (Fig. 6B). These observations were confirmed by quantification of the cellular Hsp27 mRNA level (Fig. 6C). Under these conditions, knockdown of Hsp27 was associated with a significant reduction of the amounts of viral Cap protein (Fig. 6B and C). As expected, similar results were observed in virus titer detection (Fig. 6D). These findings collectively confirm that specific inhibition of Hsp27 synthesis leads to reduction of viral protein levels.

3.7. Over-expression of Hsp27 enhanced PCV2 replicative activity To address our hypothesis that Hsp27 expression enhanced PCV2 replication, a recombinant plasmid pcDNA3.1-Hsp27-Flag was transferred into PK-15 cells. At 24 h after infected with PCV2, the cells were collected and assayed for Cap protein by immunoblotting and viral DNA by real-time PCR. Compared to those in both empty plasmid treated and PCV2 control groups, the viral DNA content in pcDNA3.1-Hsp27-Flag plasmid treated

group was increased notably (Fig. 7B) (P < 0.01). Western blotting results showed that an increase in Cap protein synthesis in pcDNA3.1-Hsp27-Flag plasmid group was observed compared to those in the control groups (Fig. 7A). Meanwhile, transfection of pcDNA3.1-Hsp27-Flag plasmid results in an increase of viral production significantly (Fig. 7C) (P < 0.05). It suggested that Hsp27 had a positive effect on PCV2 infection.

4. Discussion Porcine circovirus type 2 (PCV2) has been identified as the etiologic agent which causing postweaning multisystemic wasting syndrome widely spread in swine farms in the world. But the pathogenesis of PCV2 has not been elucidated completely. Heat shock proteins (Hsps) are involved in many viral life cycle activities, such as transcription, cellular transformation, viral genome replication, and increased virion assembly (Sullivan and Pipas, 2001). Among these, heat shock protein 27 (Hsp27), which belongs to a family of small heat shock proteins, affects protein assembly, participates in protein degradation, and prevents caspase-independent apoptosis (Ciocca et al., 1993; Mosser and Morimoto, 2004). It has reported that Hsp27 is involved in a number of viral infections, including herpes simplex virus type 1 (HSV-1), human adenovirus type 37 (HAdV-D37) and hepatitis C virus (Choi et al., 2004; Mathew et al.,

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Fig. 7. Effect of recombinant plasmid-mediated Hsp27 expression on PCV2 replication. PK-15 cells were infected with PCV2 after pcDNA3.1-Hsp27-Flag plasmid transfection. (A) Cells were lysed and cell extracts were analyzed by Western blot using anti-Hsp27, anti-Flag, anti-Cap, and anti-␤ actin antibodies. The level of relative proteins were quantified by immunoblot scanning and normalized with respect to the amount of ␤-actin (lower panel). (B) PCV2-infected PK-15 cells after recombinant plasmid transfection were assayed for viral DNA copy number by real-time PCR. (C) The cell cultures were harvested and PCV2 titers were detected as TCID50 .

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2009; Rajaiya et al., 2012). In this study, we found that Hsp27 could be up-regulated and co-localized in the nucleus with viral Cap protein during PCV2 infection. Suppression of Hsp27 with RNA interference could decrease significantly the virus replication. Meanwhile, over-expression of Hsp27 enhanced PCV2 production. It indicated that Hsp27 play important role in PCV2 replication in vitro. Hsps can be divided into 5 different types, Hsp100, Hsp90, Hsp70, Hsp60 and small Hsps, based on the molecular weight (Mw). In contrast to classic chaperones, such as Hsp70, that assist in protein folding (Young et al., 2004), Hsp27 and Hsp90 have auxiliary functions that are regulated by cochaperones and oligomeric state, respectively. The Hsp27 chaperone performs diverse functions, depending on its oligomeric and phosphorylation states (Arrigo, 2001). During times of stress, phosphorylation of Hsp27 by the mitogen-activated protein kinase, p38 occurs at three residues (Ser15, Ser78, and Ser82) (Landry et al., 1992). Data from the current study show that Hsp27 is phosphorylated at two residues (Ser15 and 78) during PCV2 infection. Viral infection stimulates accumulation of a phospho-reactive species of Hsp27, which mainly exists in the nuclear zone. Mathew and colleagues (Mathew et al., 2009) showed that treatment with chemical inhibitors blocking p38 activation results in reduced HSV-1 titers in tissue culture. In a previous report, Li and colleagues (Wei et al., 2009) showed that JNK1/2 and p38 MAPK pathways are activated during the course of PCV2 infection in PK15 cells. Moreover, activation of these two MAPK pathways is required for active replication of PCV2. In this study, inhibition of Hsp27 phosphorylation by specific chemical inhibitors and down-regulation of all forms of Hsp27 via RNA interference led to significantly reduced intracellular levels

of viral proteins and viral progeny production. In order to determine whether Hsp27 was essential to sustain a high level of virus replication, we employed recombinant plasmid-mediated Hsp27 gene expression. The results showed that plasmid-mediated gene delivery specifically increased the intracellular Hsp27 levels in PCV2-infected cells and resulted in significant increases in virion production, compared to those in the control groups. It indicated that Hsp27 is required for virus production in tissue culture. In addition, we also found that Hsp90 could be increased in the PCV2infected PK-15 cells. The role of Hsp90 in PCV2 replication should be further investigation. As we known, not all pigs infected with PCV2 will develop PMWS. Even if PMWS occurs, the severity of the disease differs at different pig farms. Infection of pigs with PCV2 and other infectious/noninfectious triggers are required for PMWS to develop (Grau-Roma et al., 2011; Patterson and Opriessnig, 2010). In pig farm, we usually observed that many piglets showed PCVAD in the season of high temperature. Previously, we demonstrated that heat-stress has a positive regulatory effect on PCV2 infection (Liu et al., 2013b). Hsp27 also could be induced by heat shock response, and its role in vivo toward PCV2 infection would be investigated in the future. In order to elucidate the molecular mechanisms involved in PCV2 infection of host cells, several earlier studies have analyzed the interplay between PCV2 and host cells using proteomics analysis, which includes interactions of PCV2 and PK15 cells (Fan et al., 2012; Zhang et al., 2009), porcine alveolar macrophages (PAMs) (Cheng et al., 2012; Liu et al., 2013a) and inguinal lymph nodes of piglets inoculated with PCV2 (Ramirez-Boo et al., 2011). Their results indicated that Hsp27 was downregulated at 72 hpi in

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PCV2-infected PK-15 cells (Zhang et al., 2009). However, in this study, Hsp27 was up-regulated in PCV2-infected PK-15 cells in the later stages at 48 and 96 h post-infection. According to this study, the reason for this observation might be due to the heterogeneity of the cell line, which being a highly permissive PK-15 cells clone for PCV2 replication. However, the difference between highly permissive PK-15 cells and normal PK-15 cells should be studied in the future. In summary, novel information on how the host small chaperone, Hsp27, positively or negatively influences virus infection may not only provide useful details about the auxiliary roles of these essential cellular proteins, but also reveal novel antiviral targets and facilitate the development of antioxidant modalities to treat viral disease. Acknowledgments This work was supported by the special fund for Agroscientific Research in the public interest (201003060-4, 201203039), the Jiangsu Province Science and Technology Support Program (BE2012368), Key Grant of Ministry of Education of China (20120097110043), the China Agricultural Research System Foundation (CARS-36), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Allan, G., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Botner, A., Charreyre, C., 1998. Novel porcine circoviruses from pigs with wasting disease syndromes. Vet. Rec. 142 (17), 467–468. Arrigo, A.P., 2001. Hsp27: novel regulator of intracellular redox state. IUBMB Life 52 (6), 303–307. Bolin, S.R., Stoffregen, W.C., Nayar, G.P., Hamel, A.L., 2001. Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus. J. Vet. Diagn. Invest. 13 (3), 185–194. Cheng, S., Zhang, M., Li, W., Wang, Y., Liu, Y., He, Q., 2012. Proteomic analysis of porcine alveolar macrophages infected with porcine circovirus type 2. J. Proteomics 75 (11), 3258–3269. Choi, Y.W., Tan, Y.J., Lim, S.G., Hong, W., Goh, P.Y., 2004. Proteomic approach identifies HSP27 as an interacting partner of the hepatitis C virus NS5A protein. Biochem. Biophys. Res. Commun. 318 (2), 514–519. Ciocca, D.R., Oesterreich, S., Chamness, G.C., McGuire, W.L., Fuqua, S.A., 1993. Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review. J. Natl. Cancer Inst. 85 (19), 1558–1570. Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G., Willson, P., Strokappe, J., Martin, K., McNeilly, F., Meehan, B., Todd, D., Haines, D., 1998. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can. Vet. J. 39 (1), 44–51. Fan, H.Y., Ye, Y., Luo, Y.W., Tong, T.Z., Yan, G.R., Liao, M., 2012. Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals protein and pathway regulation in porcine circovirus Type 2 infected PK-15 cells. J. Proteome Res. 11 (2), 995–1008. Feng, Z., Jiang, P., Wang, X., Li, Y., Jiang, W., 2008. Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo. Antiviral Res. 77 (3), 186–194. Grau-Roma, L., Fraile, L., Segales, J., 2011. Recent advances in the epidemiology, diagnosis and control of diseases caused by porcine circovirus type 2. Vet. J. 187 (1), 23–32.

Krakowka, S., Ellis, J.A., Meehan, B., Kennedy, S., McNeilly, F., Allan, G., 2000. Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet. Pathol. 37 (3), 254–263. Landry, J., Lambert, H., Zhou, M., Lavoie, J.N., Hickey, E., Weber, L.A., Anderson, C.W., 1992. Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. J. Biol. Chem. 267 (2), 794–803. Lee, G., Han, D., Song, J.Y., Lee, Y.S., Kang, K.S., Yoon, S., 2010. Genomic expression profiling in lymph nodes with lymphoid depletion from porcine circovirus 2infected pigs. J. Gen. Virol. 91 (Pt 10), 2585–2591. Li, G., Jiang, P., Li, Y., Wang, X., Huang, J., Bai, J., Cao, J., Wu, B., Chen, N., Zeshan, B., 2009. Inhibition of porcine reproductive and respiratory syndrome virus replication by adenovirus-mediated RNA interference both in porcine alveolar macrophages and swine. Antiviral Res. 82 (3), 157–165. Liu, J., Bai, J., Lu, Q., Zhang, L., Jiang, Z., Michal, J.J., He, Q., Jiang, P., 2013a. Twodimensional liquid chromatography–tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2. J. Proteomics 79, 72–86. Liu, J., Bai, J., Zhang, L., Jiang, Z., Wang, X., Li, Y., Jiang, P., 2013b. Hsp70 positively regulates porcine circovirus type 2 replication in vitro. Virology 447 (1–2), 52–62. Liu, Q., Tikoo, S.K., Babiuk, L.A., 2001. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2. Virology 285 (1), 91–99. Mathew, S.S., Della Selva, M.P., Burch, A.D., 2009. Modification and reorganization of the cytoprotective cellular chaperone Hsp27 during herpes simplex virus type 1 infection. J. Virol. 83 (18), 9304–9312. McNair, I., Marshall, M., McNeilly, F., Botner, A., Ladekjaer-Mikkelsen, A.S., Vincent, I., Herrmann, B., Sanchez, R., Rhodes, C., 2004. Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2. J. Vet. Diagn. Invest. 16 (2), 164–166. Mosser, D.D., Morimoto, R.I., 2004. Molecular chaperones and the stress of oncogenesis. Oncogene 23 (16), 2907–2918. Nawagitgul, P., Morozov, I., Bolin, S.R., Harms, P.A., Sorden, S.D., Paul, P.S., 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J. Gen. Virol. 81 (Pt 9), 2281–2287. Patterson, A.R., Opriessnig, T., 2010. Epidemiology and horizontal transmission of porcine circovirus type 2 (PCV2). Anim. Health Res. Rev./Conf. Res. Workers Anim. Dis. 11 (2), 217–234. Rajaiya, J., Yousuf, M.A., Singh, G., Stanish, H., Chodosh, J., 2012. Heat shock protein 27 mediated signaling in viral infection. Biochemistry 51 (28), 5695–5702. Ramirez-Boo, M., Nunez, E., Jorge, I., Navarro, P., Fernandes, L.T., Segales, J., Garrido, J.J., Vazquez, J., Moreno, A., 2011. Quantitative proteomics by 2-DE, 16O/18O labelling and linear ion trap mass spectrometry analysis of lymph nodes from piglets inoculated by porcine circovirus type 2. Proteomics 11 (17), 3452–3469. Rogalla, T., Ehrnsperger, M., Preville, X., Kotlyarov, A., Lutsch, G., Ducasse, C., Paul, C., Wieske, M., Arrigo, A.P., Buchner, J., Gaestel, M., 1999. Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation. J. Biol. Chem. 274 (27), 18947–18956. Shibahara, T., Sato, K., Ishikawa, Y., Kadota, K., 2000. Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome. J. Vet. Med. Sci./Jpn. Soc. Vet. Sci. 62 (11), 1125–1131. Sullivan, C.S., Pipas, J.M., 2001. The virus-chaperone connection. Virology 287 (1), 1–8. Tischer, I., Gelderblom, H., Vettermann, W., Koch, M.A., 1982. A very small porcine virus with circular single-stranded DNA. Nature 295 (5844), 64–66. Wei, L., Zhu, Z., Wang, J., Liu, J., 2009. JNK and p38 mitogen-activated protein kinase pathways contribute to porcine circovirus type 2 infection. J. Virol. 83 (12), 6039–6047. Young, J.C., Agashe, V.R., Siegers, K., Hartl, F.U., 2004. Pathways of chaperonemediated protein folding in the cytosol. Nat. Rev. Mol. Cell Biol. 5 (10), 781–791. Zhang, X., Zhou, J., Wu, Y., Zheng, X., Ma, G., Wang, Z., Jin, Y., He, J., Yan, Y., 2009. Differential proteome analysis of host cells infected with porcine circovirus type 2. J. Proteome Res. 8 (11), 5111–5119.

Please cite this article in press as: Liu, J., et al., Heat shock protein 27 is involved in PCV2 infection in PK-15 cells. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.024

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