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Pyrithione inhibits porcine reproductive and respiratory syndrome virus replication through interfering with NF-κB and heparanase
Veterinary Microbiology 201 (2017) 231–239
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Pyrithione inhibits porcine reproductive and respiratory syndrome virus replication through interfering with NF-kB and heparanase Chunhe Guo, Zhenbang Zhu, Xiaoying Wang, Yaosheng Chen, Xiaohong Liu * State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou Higher Education Mega Center, North Third Road, Guangzhou, Guangdong 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2016 Accepted 28 January 2017
Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccination strategies provide only limited protection against PRRSV infection. Consequently, there is a need to develop new antiviral strategies. Pyrithione (PT), a zinc ionophore, is used as an antibacterial and antifungal agent, and evidence has shown that PT inhibits the replication of various RNA viruses. However, there is no data regarding its effects against PRRSV infection until now. In this study, we showed that PT strongly inhibited PRRSV replication in Marc-145 cells. Similar inhibitory effects were also found in porcine alveolar macrophages, the major target cell type of PRRSV infection in pigs in vivo. PT also attenuated virus-induced apoptosis during the late phase of infection. In addition, we provided evidence that PT caused a rapid import of extracellular zinc ions into cells, and imported Zn2+ was responsible for its antiviral property. We investigated the molecular mechanisms of PT against PRRSV and found that PT inhibited NF-kB and heparanase, producing the increased heparan sulfate expression to block the release of virus and cytokines, thus decreasing viral replication. These findings suggest that PT has the potential to the development of prophylactic and therapeutic strategies against PRRSV infection. ß 2017 Elsevier B.V. All rights reserved.
Keywords: PRRSV Pyrithione Antiviral activity.
1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant porcine infectious diseases in the pig industry worldwide. It was first reported at the end of the 1980s in North America and Canada, and is commonly known as blue-eared pig disease (Zhao et al., 2014). The disease is characterized by abortion and poor reproductive performance in pregnant sows, respiratory distress in piglets and growing pigs, and high mortality in piglets (Music and Gagnon, 2010; Yun and Lee, 2013). PRRS virus (PRRSV), the etiological agent of the disease, belongs to the Arteriviridae family, together with equine arteritis virus, lactate dehydrogenaseelevating virus, and simian hemorrhagic fever virus. Like other arteriviruses, the viral genome is an enveloped, single, positivesense RNA strand of approximately 15 kb with a 50 -untranslated region (UTR), nine open reading frames (ORFs 1a, 1b, 2a, 2b, and
* Corresponding author. Tel.: +86 20 39332788; fax: +86 20 39332940. E-mail addresses: [email protected] (C. Guo), [email protected] (Z. Zhu), [email protected] (X. Wang), [email protected] (Y. Chen), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.vetmic.2017.01.033 0378-1135/ß 2017 Elsevier B.V. All rights reserved.
3–7) and a 30 -UTR (Fang and Snijder, 2010). According to its genetic diversity and geographic distribution, PRRSV can be broadly divided into two distinct genotypes with clear genetic and antigenic differences: the European type Lelystad strain (type 1) and the North American type VR-2332 strain (type 2), both of which share about 60% nucleotide sequence identity (Murtaugh et al., 2010; Shi et al., 2010). Pigs persistently infected with PRRSV develop viremia, delayed onset and low titer of neutralizing antibodies and reduced cellmediated immunity, they may shed virus for several months (Hu et al., 2012). Important features of PRRSV are its extreme genetic, antigenic, and immunobiological variability (Guo et al., 2014). Consequently, PRRSV remains the great challenge to swine industry, which highlights the need for novel and effective strategies to control PRRSV transmission (Guo et al., 2015a). Zinc ions are involved in many different cellular processes and have proven crucial for the proper folding and activity of various cellular enzymes and transcription factors. Maintaining the appropriate Zn2+ concentration is critical for cell survival since both increased and decreased Zn2+ levels can trigger apoptosis in a variety of cell types (te Velthuis et al., 2010). It is also an important
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cofactor for numerous viral proteins (te Velthuis et al., 2010). Previous studies have shown that polyprotein processing and protease activity are inhibited by Zn2+ in cells infected with human rhinovirus and coxsackievirus B3 (te Velthuis et al., 2010). Pyrithione (PT), a zinc ionophore, commonly acts as a centrosymmetric dimmer to increase the intracellular Zn2+ concentration via its oxygen and sulfur centers (Qiu et al., 2013). It is approved by Food and Drug Administration as worldwide, over-the-counter, topical antimicrobials for psoriasis and epidermal hyperplasia. PT is used as an antibacterial and antifungal agent, and evidence has shown that PT inhibits the replication of various RNA viruses, including influenza virus, respiratory syncytial virus and several picornaviruses (te Velthuis et al., 2010). In this study, we demonstrated that PT exhibited potent antiviral activity against PRRSV infection both in Marc-145 cells and pocine alveolar macrophage (PAMs). Mechanistic study showed that PT inhibited PRRSV replication via interfering with NF-kB and heparanase. 2. Materials and methods 2.1. Cells and viruses Porcine alveolar macrophages (PAMs) were obtained with lung lavage from the lungs of 3–8-week-old PRRSV-negative piglets (Guo et al., 2015b) and cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAA, Pasching, Austria), 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate at 37 8C in 5% CO2. All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Marc-145 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) containing 10% of FBS. Highly pathogenic PRRSV (HP-PRRSV) strain Li11 was propagated and titrated in PAMs or Marc-145 cells and used throughout the study. 2.2. Cell viability assay The viability of Marc-145 cells and PAMs after PT treatment was determined using the alamarBlue1 assay (Invitrogen, CA, USA) which is used to establish relative cytotoxicity of agents. Briefly, a series of concentrations of PT, diluted in DMEM or RPMI-1640, was applied to 60%–70% confluent Marc-145 cells or PAMs. After incubation for 48 h at 37 8C in a 5% CO2 atmosphere, 10 ml of alamarBlue1 was added and the cultures were incubated for another 3 h. Fluorescence intensity was detected at 570 nm excitation and 590 nm emission wavelengths and directly proportional to the number of living cells in culture. 2.3. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR)
After incubating for the indicated time, cells were rinsed with phosphate-buffered saline (PBS) three times and blocked with 1% Bovine serum albumin (BSA) in PBS for 30 min, then incubated with anti-PRRSV N protein mAb (SDOW17, Jeno Biotech Inc, Republic of Korea), anti-NF-kB p65 mAb (Cell Signal Technolgy (CST), MA, USA), anti-cleaved caspase-3 mAb (CST) or anti-heparan sulfate (HS) mAb (abcam, MA, USA) and followed by Alexa Fluor1 555-conjugated anti-mouse IgG secondary antibody for 2 h. After three washes in PBS, cell nuclei were stained with Hoechst dye 33258 (Sigma, MO, USA) and detected using fluorescence microscopy (Carl Zeiss, Jena, Germany). 2.5. Western blot analysis Marc-145 cells or PAMs cultured in six-well plates were harvested in lysis buffer (Beyotime, Jiangsu, China) containing a cocktail of protease inhibitors (Roche) and separated by 10% SDSPAGE, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After the membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris [pH 7.5], 150 mM NaCl, 0.5% Tween 20) for 2 h at 37 8C, they were rinsed and incubated with anti-PRRSV N protein mAb (SDOW17), antiheparanase mAb (abcam), anti-NF-kB p65 mAb (CST), antiglyceraldehyde phosphate dehydrogenase (GAPDH) mAb (CST), horseradish-peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (CST). Protein bands were visualized with ECL Plus chemiluminescence reagent (Pierce, Rockford, USA). 2.6. Virus titration assay In order to analyze the growth of PRRSV, the viral supernatants from cell cultures were collected at indicated time points after virus infection. Virus yields were measured by the 50% tissue culture infected dose (TCID50) assay using the Reed–Muench method. Briefly, cells were seeded in 96-well plates before virus infection. Viral supernatants were prepared by 10-fold serial dilution and 100 ml of dilutions were added per well in eight replicates. Virus titers were calculated 4–5 days post infection. 2.7. Apoptosis assay Apoptosis was evaluated by an Annexin V-FITC/Propidium Iodide (PI) assay (BD Biosciences Pharmingen, CA, USA) according to the manufacturer’s protocol. Marc-145 cells were mockinfected or infected with PRRSV at an multiplicity of infection (MOI) of 0.1 in the presence of PT for 36 h, then resuspended in 1 Binding Buffer after washed twice with cold PBS. Finally cells were stained with FITC Annexin V and PI for 15 min at RT in the dark and analyzed by flow cytometry (BD FACSCalibur, USA) within 1 h. 2.8. Antiviral assay
Total RNA was isolated from Marc-145 cells or PAMs using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions, and subjected to qRT-PCR analysis. cDNA was synthesized using reverse transcription kit (Promega, Madison, WI). SYBR Green (TaKaRa, Osaka, Japan) real-time PCR was performed using Light-Cycler 480 PCR system (Roche, Basel, Switzerland). Relative quantities of mRNA accumulation were D evaluated using the 2 Ct method. Primers will be made available upon request.
Marc-145 cells or PAMs were seeded in six-well plates and grown to 60%–70% confluence at 37 8C in 5% CO2, then inoculated with PRRSV at an MOI of 0.1 in the presence of different concentrations of PT. After incubation for 24 h, the supernatants were collected to titrate the viral yields, and the viral titers were determined as TCID50 (Delogu et al., 2011). The cells were harvested for IFA, qRT-PCR, and western blot analysis. 2.9. Viral release assay
2.4. Immunofluorescence assay (IFA) Cells were seeded onto coverslips, and then fixed with 4% paraformaldehyde and permeabilized at room temperature (RT).
Marc-145 cells were washed three times with PBS and then treated with PT at 37 8C for 4 h after challenged with PRRSV (MOI = 0.1) at 37 8C for 24 h. The cell culture supernatants were
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collected to titrate the viral yields and to quantify viral particles using TCID50 and qRT-PCR methods. 2.10. Cell fractionation Marc-145 cells were fractionated using a Nuclear/Cytosol Fractionation Kit (BioVision, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were washed twice with icecold PBS, and transferred to 1.5 ml Eppendorf tubes, and then centrifuged at 600 g for 5 min at 4 8C. The pellet was resuspended in cold Cytosol Extraction Buffer A and B. After centrifugation at maximal speed for 5 min, the cytoplasmic extract fraction was transferred to a clean pre-chilled tube. For the nuclear extract, the pellet was resuspended in cold Nuclear Extraction Buffer A and centrifuged at full speed for 10 min. The extracted nuclear and cytoplasmic protein fractions were prepared for western blot analysis. 2.11. Statistical analysis All experiments were performed with at least three independent replicates. All data were presented as means standard errors (SE). Statistics analysis was performed by SPSS 16.0 using Student’s ttest and one-way ANOVA. Differences with P values <0.05 were considered significant.
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3. Results 3.1. PT strongly inhibits PRRSV infection and replication in Marc-145 cells To evaluate the in vitro antiviral activity of PT against PRRSV infection, cytotoxicity assay was essential for the initial phase of antiviral drug development. We first investigated its cytotoxicity on Marc-145 cells using alamarBlue1. As shown in Fig. 1A, no significant cytotoxicity was observed up to 80 mg/ml PT. Therefore, we performed all subsequent experiments with PT at a concentration no higher than 80 mg/ml in Marc-145 cells. Next cytopathic effects (CPE) observation, IFA and virus titers assays were performed to determine the effect of PT on PRRSV infection. PT (15 mg/ml) notably abrogated the PRRSV-induced CPE at 72 h post infection (hpi) in Marc-145 cells infected with PRRSV at an MOI of 0.1 (Fig. 1B). The specificity of CPE was further confirmed by IFA with anti-PRRSV N protein mAb at 24 hpi. Upon PT treatment, N-specific staining was notably reduced in a dosedependent manner in Marc-145 cells compared to the control without PT treatment (Fig. 1C), indicating that infection and the spread of the virus to the neighboring cells are blocked by PT. Consistent with these findings, the production of viral progeny was significantly reduced at 24 hpi by PT, in a dose-dependent manner (Fig. 1D).
Fig. 1. Antiviral activity of PT against PRRSV infection in Marc-145 cells. (A) Potential cytotoxicity of PT against Marc-145 cells was detected with alamarBlue1. A series of concentrations of PT was applied to 60%–70% confluent cells for 48 h, and then 10 ml of alamarBlue1 was added. (B) Cells were mock infected or infected with PRRSV at an multiplicity of infection (MOI) of 0.1 in the presence or absence of PT (15 mg/ml). Virus-produced cytopathic effects (CPE) were observed by bright-field microscopy at 72 h post infection (hpi). (C) Cells were fixed with 4% paraformaldehyde in the presence or absence of different concentrations of PT at 24 hpi, then analyzed by IFA using antiPRRSV N protein mAb (red). Bar, 200 mm. (D) Virus titers in virus-infected cells (MOI = 0.1) were determined followed by treatment with or without PT at 24 hpi. (E and F) Cells were inoculated with PRRSV (MOI = 0.1) in the presence or absence of different concentrations of PT for 24 h, the transcript (E) and protein (F) levels of PRRSV N gene were determined. Expression of GAPDH was shown as a loading control. (G and H) To evaluate whether PT can inhibit the level of N protein both in the nuclear and cytoplasmic fractions of PRRSV-infected cells. The amount of N protein both in the cytoplasm (G) and nucleus (H) in cells treated with or without PT was determined at 24 hpi. GAPDH and Histone H3 were used as cytoplasmic and nuclear controls, respectively. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
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Since viral RNA transcription and protein translation are functionally connected in PRRSV replication (Lee and Lee, 2010), we attempted to assess the transcript and protein levels of PRRSV N gene in the presence or absence of PT at 24 hpi in Marc-145 cells. As shown in Fig. 1E, the cells treated with PT showed a significant reduction in the transcript level of PRRSV N gene in a dosedependent manner compared to those without PT treatment. Consistently, the translation level of the viral N protein was decreased significantly by PT in comparison to the control (Fig. 1F). Since the N protein of PRRSV is found not only in the cytoplasm but also in the nucleus of virus-infected cells (Lee and Lee, 2016), we attempted to evaluate whether PT can inhibit the level of N protein both in the nuclear and cytoplasmic fractions of PRRSV-infected cells. As shown in Fig. 1G and H, the amount of N protein both in the nucleus and cytoplasm in PRRSV-infected cells was notably diminished in the presence of PT at 24 hpi. Taken together, these data indicate that PT exerts an inhibitory effect on PRRSV infection in Marc-145 cells. 3.2. PT inhibits PRRSV replication in PAMs Since PT effectively inhibited PRRSV infection and replication in Marc-145 cells, we investigated whether PT also inhibits PRRSV replication in PAMs, the major target cell type of PRRSV infection in pigs in vivo. We initially performed cell viability assay on PAMs according to the methods described in ‘‘Materials and methods’’. PT was noncytotoxic up to 60 mg/ml after incubation for 48 h (Fig. 2A). Next we evaluated its effect on PRRSV replication in PAMs. Consistent with the findings obtained with Marc-145 cells, PT (15 mg/ml) caused significant reductions in the viral N protein at the transcript and protein levels and in the viral titers at 24 hpi,
Fig. 2. PT inhibits PRRSV replication in PAMs. (A) Cytotoxic activity of PT against PAMs was measured using the alamarBlue1. PAMs were incubated with various concentrations of PT for 48 h. (B–D) PAMs were infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml) for 24 h. The viral titers in the supernatants (B), viral RNA transcription (C) and protein (D) levels were analyzed. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
Fig. 3. PT blocks PRRSV-induced apoptosis during the late phase of infection. (A) Marc-145 cells were mock-infected or infected with PRRSV at an MOI of 0.1 in the presence or absence of PT (15 mg/ml). After incubation for 36 h, cells were detected by IFA with anti-cleaved caspase-3 mAb (red). Bar, 200 mm. (B) Cells were mock-infected or infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml) for 36 h, then resuspended in 1 Binding Buffer after washed twice with cold PBS. Finally cells were stained with FITC Annexin V and PI for 15 min and analyzed by flow cytometry (BD FACSCalibur, USA) within 1 h. Q1, necrotic or another cell population that was FITC Annexin V-negative and PI-positive; Q2, end stage apoptotic or a dead cell population that was FITC Annexin V- and PI-positive; Q3, an early apoptotic cell population that was FITC Annexin V positive and PI-negative; Q4, a viable cell population that was not undergoing apoptosis and was both FITC Annexin V- and PI-negative.
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compared with those of cells without PT treatment (Fig. 2B–D). Taken together, PT inhibits PRRSV replication in PAMs as well. 3.3. PT blocks PRRSV-induced apoptosis during the late phase of infection Previous studies have shown that PRRSV stimulates antiapoptotic pathways in Marc-145 cells and PAMs early in infection and that PRRSV-infected cells die from apoptosis late in infection (Liu et al., 2015). To investigate whether PT can block virusinduced apoptosis during the late phase of infection, IFA with anticleaved caspase-3 mAb was performed. As shown in Fig. 3A, upon PT treatment (15 mg/ml), cleaved caspase-3-specific staining (red) was notably reduced in Marc-145 cells at 48 hpi compared to the control without PT treatment. Further quantification of apoptotic cells was performed with flow cytometry. The results showed that the apoptosis of PRRSV-infected cells were significantly decreased after treatment with PT at 48 hpi. Together, these data demonstrate that PT blocks PRRSV-induced apoptosis during the late phase of infection, which might contribute to its inhibition of PRRSV infection. 3.4. PT leads to a rapid import of extracellular zinc ions into cells To directly visualize the ability of PT to increase the intracellular pool of labile Zn2+, fluorescence microscopy with specific zinc indicators was employed. FluoZinTM-3 (Invitrogen, CA, USA) is suitable for detection of Zn2+ concentrations in the 1–100 nM range and it has shown to be a Zn2+-sensitive and Zn2+-specific fluorescent probe that has no specific cellular localization (Krenn et al., 2009). As shown in Fig. 4A and B, in cells loaded with
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FluoZinTM-3, intracellular fluorescence was minimal due to a very low level of free Zn2+. After the application of the zinc ionophore PT in cells, the fluorescence was significantly increased in dose- and time-dependent manners (Fig. 4A and B), indicating that the intracellular labile ‘‘chelatable’’ zinc level is elevated. 3.5. Zn2+ is responsible for the antiviral property of PT Since Zn2+ plays a vital role in the bioactivities of PT (Qiu et al., 2013), we investigated the importance of Zn2+ in PT inhibition of PRRSV replication. The specific role of Zn2+ was illustrated by the treatment of metal ion chelator EDTA-Mg (Sigma) and N,N,N0 ,N0 tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, Sigma). In the presence of EDTA-Mg (10 mM), PT (15 mg/ml) inhibition of the transcript and protein levels of viral N protein was partially abrogated since Zn2+ in the culture medium competes for binding EDTA-Mg to form higher affinity EDTA-Zn, thus depleting the free Zn2+ (Fig. 4C and D). Consistently, TPEN (3 mg/ml) was capable of mitigating the antiviral property of PT against PRRSV replication (Fig. 4E). These data indicates that the availability of Zn2+ is a prerequisite for the anti-PRRSV activity of PT. 3.6. PT inhibition of NF-kB activation might contribute to its antiPRRSV activity It is well known that PRRSV infection activates NF-kB pathway, an essential step for effective viral gene expression and replication (Zhang et al., 2016). To investigate whether PT can inhibit PRRSVinduced NF-kB activation in Marc-145 cells, confocal microscopy was performed. As shown in Fig. 5A, in PRRSV-infected cells, nuclear accumulation of endogenous p65 (red) was observed,
Fig. 4. PT increases the intracellular pool of labile Zn2+ which is responsible for its antiviral activity. (A and B) Cells were loaded with FluoZinTM-3 acetoxymethyl (AM) ester (Invitrogen, CA, USA) in PBS for 15 min, and incubated for a further 30 min in growth medium to allow complete de-esterification of intracellular AM esters. PT was then added to the medium for the indicated concentrations (A) and times (B). Fluorescence was monitored in a live-cell microscope system (Carl Zeiss, Jena, Germany) using fluorescein isothiocyanate settings for FluoZinTM-3. Bar, 200 mm. (C and D) Cells were infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml) and EDTA-Mg (10 mM) for 24 h, the transcript (C) and protein (D) levels of PRRSV N gene were determined by qRT-PCR and western blot analysis. (E) The transcript level of PRRSV N gene in infected cells in the presence or absence of PT (15 mg/ml) and TPEN (3 mg/ml) was analyzed at 24 hpi. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
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Fig. 5. PT inhibition of NF-kB activation might contribute to its anti-PRRSV activity. (A) Marc-145 cells were mock-infected or infected with PRRSV at an MOI of 0.1 in the presence or absence of PT (15 mg/ml) for 24 h, and then fixed with 4% paraformaldehyde and analyzed by IFA using anti-NF-kB p65 mAb (CST, MA, USA) (red). Bar, 25 mm. (B) To further confirm that PT can inhibit PRRSV-induced NF-kB activation, a cell fractionation assay in cells treated with or without PT (15 mg/ml) was performed at 24 hpi. GAPDH and Histone H3 were used as cytoplasmic and nuclear controls, respectively. (C–E) Virus-infected or uninfected PAMs were cultured in the presence of PT (15 mg/ml) for 18 h. The expression of IFN-a (C), IFN-b (D) and TNF-a (E) was analyzed by qRT-PCR. (F and G) Marc-145 cells were mock-infected or infected with PRRSV (MOI = 0.1) in the presence of PT (15 mg/ml) for 18 h. qRT-PCR was conducted to evaluate the expression of IL-6 (F) and TNF-a (G). Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
indicating that NF-kB is activated as expected. However, in virusinfected cells treated with PT (15 mg/ml), p65 mainly existed in cytoplasm, similar to the results of uninfected mock-treated cells (top panel). Consistent with above data, we performed a cell fractionation assay and found that PT (15 mg/ml) attenuated PRRSV-mediated p65 nuclear translocation (Fig. 5B), further confirming that PT inhibits PRRSV-induced NF-kB activation. NF-kB is an important transcription factor for cytokines production. PRRSV infection activates NF-kB and subsequent production of inflammatory cytokines. Whether PT can inhibit PRRSV-induced upregulation of cytokines is also investigated. PT (15 mg/ml) strongly inhibited the expression of IFN-a, IFN-b and TNF-a induced by PRRSV in PAMs (Fig. 5C–E). Consistently, the upregulation of IL-6 and TNF-a expression was significantly reduced by PT in PRRSV-infected Marc-145 cells (Fig. 5F and G). These results demonstrate that PT can interfere with the
production of NF-kB-mediated cytokines caused by PRRSV infection. 3.7. PT inhibits PRRSV-induced heparanase Previous studies have shown that heparanase is implicated in the regulation of various physiological and pathological processes (Rivara et al., 2016). Upon herpes simplex virus-1 (HSV-1) infection, heparanase is upregulated through NF-kB pathway and translocated to the cell surface to facilitate viral release (Hadigal et al., 2015). Initially, we investigated whether PRRSV can induce heparanase expression in Marc-145 cells. Western blot and qRT-PCR analysis revealed that expression of heparanase was significantly greater at 36 hpi than in uninfected cells (Fig. 6A and B). Next we evaluated the effect of PT on heparanase induced by PRRSV in Marc-145 cells. As shown in Fig. 6C and D, in the presence
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of PT (15 mg/ml), the transcript and protein levels of heparanase were significantly reduced at 36 hpi. Collectively, these data suggest that PT is able to inhibit PRRSV-induced heparanase. 3.8. PT inhibition of HS loss from cell surface after infection might contribute to its capacity of blocking viral release
Fig. 6. PT inhibits PRRSV-induced heparanase. (A and B) Cells were mock-infected or infected with PRRSV at an MOI of 0.1 for 36 h, and then harvested for the transcript (A) and protein (B) levels of heparanase by qRT-PCR and western blot analysis. (C and D) Virus-infected cells were cultured in the presence or absence of PT (15 mg/ ml) for 36 h. Heparanase transcription (C) and protein (D) levels were analyzed. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
HS is a ubiquitously expressed glycosaminoglycan that is used as a docking site for initial attachment by many viruses such as dengue virus, foot-and-mouth disease virus and PRRSV (Hadigal et al., 2015). However, during a productive infection, the HS moieties on parent cells can trap newly exiting viral progenies and block viral release (Hadigal et al., 2015). To determine whether PT can inhibit the loss of HS from cell surface after PRRSV infection in Marc-145 cells, qRT-PCR and confocal microscopy with anti-HS mAb assays were performed. As shown in Fig. 7A and B, a dramatic decrease in HS was seen during later stage of infection (36 hpi) in Marc-145 cells compared with that in uninfected mock-treated cells. However, in the presence of PT (15 mg/ml), HS from the surface of PRRSV-infected cells was significantly increased at 36 hpi, indicating that PT can inhibit HS degradation during later stage of PRRSV infection. Since HS is able to block viral release during a productive infection, we investigated whether PT can inhibit PRRSV release in Marc-145 cells. Our results showed that significantly fewer infectious viral particles were released into the supernatant by cells treated with PT (15 mg/ml) for 4 h than by untreated cells at 36 hpi (Fig. 7C). The viral titers in the supernatants showed a similar pattern after treatment with PT (15 mg/ml) for 4 h (Fig. 7D). Taken together, these data indicate that PT inhibits HS degradation
Fig. 7. PT inhibition of HS degradation might contribute to its capacity of blocking viral release. (A) Cells were fixed with 4% paraformaldehyde in the presence or absence of PT (15 mg/ml) at 36 hpi, then analyzed by IFA using anti-HS mAb (red). Bar, 25 mm. (B) Cells were mock-infected or infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml). After incubation for 36 h, cells were collected to determine RNA transcription of HS. (C and D) Cells were infected with PRRSV at an MOI of 0.1 for 36 h, and then treated with PT (15 mg/ml) for 4 h. The supernatants were harvested to measure extracellular viral particles (C) and viral titers (D) using qRT-PCR and TCID50, respectively. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
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Fig. 8. Schematic model of anti-PRRSV activity of PT. During the productive period of infection, PRRSV activates NF-kB to promote HPSE mRNA expression, then the cytoplastic heparanase are translocated to the cell surface and exert the enzymatic activity to process the ECM, resulting in the release of virus and cytokines attached to fragments of ECM-resident HS. As an inhibitor of NF-kB and HPSE, PT can inhibit this effect.
induced by PRRSV during later stage of infection, which might contribute to its inhibition of viral release. Heparanase is the only known mammalian endoglycosidase that cleaves HS, facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines in a number of physiological and pathological settings (Yang et al., 2015). Previous studies have shown that HSV-infected cells upregulate heparanase through NF-kB, which is then translocated to the cell surface, producing the decreased HS expression to allow viral exit (Hadigal et al., 2015). Based in our results, the inhibitory mechanisms of PT may be illuminated in the model (Fig. 8). During the productive period of infection, PRRSV activates NF-kB to promote HPSE mRNA expression, then the cytoplastic heparanase are released to the extracellular space and exert the enzymatic activity to process the ECM, resulting in release of virus and cytokines attached to fragments of ECMresident HS. As an inhibitor of NF-kB and heparanase, PT can inhibit this effect. 4. Discussion PRRSV first emerged in the late 1980s in North America, and subsequently in Europe, causing huge economic losses. It has since spread across the globe, inducing characteristic severe reproductive failure in female pigs and respiratory tract illness in piglets. PRRSV primarily infects PAMs and is characterized by the high rate of mutation and recombination (Deaton et al., 2014). Moreover, it causes the delayed appearance and low titer of neutralizing antibodies and initiates a comprehensive campaign against the innate immune response (Fang et al., 2012; Sun et al., 2010). Therefore, there is an urgent need for developing new antiviral strategies against PRRSV infection. PT, known to be a zinc ionophore that leads to a rapid increase in intracellular zinc levels, is effective against several pathogens such as human rhinovirus, coxsackievirus, mengovirus and HSV (Qiu et al., 2013).
In the current study, we showed that PT exhibited potent antiviral activity against PRRSV infection and replication in vitro at noncytotoxic concentrations (Figs. 1 and 2). Mechanistic study showed that PT inhibited the expression of heparanase via interfering with NF-kB activation, and then blocked the loss of HS from the cell surface, thus inhibited the release of virus and cytokines (Fig. 8). We provided evidence that the basis of the antiviral activity was dependent upon the availability of zinc ions as this inhibitory activity could be blocked by the addition of Zn2+ chelators, such as EDTA-Mg and TPEN (Fig. 4). The import of extracellular zinc ions is a key feature of the antiviral property of PT, which is consistent with previous reports (Krenn et al., 2009). The PRRSV N protein, the most abundant viral protein expressed in infected cells, blocks host protein synthesis due to the high concentration in the nucleolus (Jourdan et al., 2012). Moreover, the N protein may impact transcriptional regulation in infected cells owing to its interacting with transcriptional regulators (Dokland, 2010). In this study, our data demonstrated that PT effectively decreased the transcription and translation levels of the viral N protein (Figs. 1 and 2). Moreover, PT exhibited robust anti-PRRSV activity via multiple pathways including inhibition of virus progeny production, virus-induced apoptosis during the late phase of infection, and viral particle release (Figs. 1, 3 and 7). Since Marc-145 cells are not of porcine origin but are monkey cells (Zhang et al., 2013), we also examined the antiviral activity of PT in PAMs, which are known to be the primary host cell target for PRRSV infection in vivo. Consistent with the data obtained with Marc-145 cells, PT strongly inhibited virus replication in PAMs (Fig. 2), indicating that it might be an effective inhibitor of PRRSV infection in vivo. HS is a key component of the ECM and is used by many viruses for initial attachment to target cells. As the only known enzyme capable to degrade HS, heparanase mediates the release of HSbound cytokines, growth factors and viruses (Yang et al., 2015). Previous studies have demonstrated that HS expression is
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increased during the initial stages of HSV-1 infection to enhance viral attachment, while a dramatic decrease in HS is seen during later stages of infection. The results indicate that heparanase is upregulated in the loss of HS late during HSV-1 infection. In this study, we showed that expression of heparanase was elevated and a dramatic decrease in HS was seen during later stage of PRRSV infection (36 hpi, Figs. 6 and 7), which is consistent with above research. This identifies a novel role for heparanase and HS in the regulation of PRRSV infection. However, further research should be undertaken to investigate the expression of HS and heparanase during the initial stages of PRRSV infection and whether viral attachment and detachment modes are regulated by the host enzyme heparanase. In physiological conditions, heparanase is expressed at high levels only in few tissues while in specific pathological conditions, it is upregulated and highly affects multiple biological processes, indicating that it might be recognized as a negative prognostic marker (Rivara et al., 2016). Heparanase is involved in many different pathological scenarios such as inflammatory diseases, angiogenesis, cancer and metastasis (Rivara et al., 2016). Therefore, heparanase has attracted considerable attention as a promising target for innovative pharmacological applications. PI-88, a potent heparanase inhibitor that is currently in human clinical trials for cancer treatment, can reduce viral release and control the host inflammatory response (Rivara et al., 2016). In this study, we demonstrated that heparanase was upregulated in PRRSV-infected cells while in the presence of PT, it was significantly decreased at 36 hpi (Fig. 6), indicating that PT inhibits viral replication via interfering with heparanase which might be a unique therapeutic target against PRRSV. In conclusion, the results presented herein demonstrate that PT exerts a potent inhibitory effect on PRRSV through interfering with NF-kB and heparanase, indicating that PT has the potential to the development of prophylactic and therapeutic strategies against PRRSV infection. Further research is required to evaluate the potential in vivo antiviral activity of PT in animal models. Competing interests The authors declare that they have no competing interests. Acknowledgements This work was supported by National Natural Science Foundation of China (31601917), Natural Science Foundation of Guangdong Province (2014A030312011) and Guangdong Sailing Program (2014YT02H042). References Deaton, M.K., Spear, A., Faaberg, K.S., Pegan, S.D., 2014. The vOTU domain of highlypathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference. Virology 454-455, 247–253. Delogu, I., Pastorino, B., Baronti, C., Nougairede, A., Bonnet, E., de Lamballerie, X., 2011. In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant. Antivir. Res. 90, 99–107. Dokland, T., 2010. The structural biology of PRRSV. Virus Res. 154, 86–97. Fang, Y., Snijder, E.J., 2010. The PRRSV replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins. Virus Res. 154, 61–76.
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Fang, Y., Treffers, E.E., Li, Y., Tas, A., Sun, Z., van der Meer, Y., de Ru, A.H., van Veelen, P.A., Atkins, J.F., Snijder, E.J., Firth, A.E., 2012. Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein. Proc. Natl. Acad. Sci. USA 109, E2920–E2928. Guo, C., Chen, L., Mo, D., Chen, Y., Liu, X., 2015a. DRACO inhibits porcine reproductive and respiratory syndrome virus replication in vitro. Arch. Virol. 160, 1239–1247. Guo, C., Cong, P., He, Z., Mo, D., Zhang, W., Chen, Y., Liu, X., 2015b. Inhibitory activity and molecular mechanism of protegrin-1 against porcine reproductive and respiratory syndrome virus in vitro. Antivir. Ther. 20, 573–582. Guo, C., Huang, Y., Cong, P., Liu, X., Chen, Y., He, Z., 2014. Cecropin P1 inhibits porcine reproductive and respiratory syndrome virus by blocking attachment. BMC Microbiol. 14, 273. Hadigal, S.R., Agelidis, A.M., Karasneh, G.A., Antoine, T.E., Yakoub, A.M., Ramani, V.C., Djalilian, A.R., Sanderson, R.D., Shukla, D., 2015. Heparanase is a host enzyme required for herpes simplex virus-1 release from cells. Nat. Commun. 6. Hu, J., Ni, Y., Dryman, B.A., Meng, X.J., Zhang, C., 2012. Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV). Vaccine 30, 2068–2074. Jourdan, S.S., Osorio, F.A., Hiscox, J.A., 2012. Biophysical characterisation of the nucleocapsid protein from a highly pathogenic porcine reproductive and respiratory syndrome virus strain. Biochem. Biophys. Res. Commun. 419, 137–141. Krenn, B.M., Gaudernak, E., Holzer, B., Lanke, K., Van Kuppeveld, F.J., Seipelt, J., 2009. Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections. J. Virol. 83, 58–64. Lee, Y.J., Lee, C., 2010. Porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. Virus Res. 152, 50–58. Lee, Y.J., Lee, C., 2016. Ivermectin inhibits porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages. Arch. Virol. 161, 257–268. Liu, X., Guo, C., Huang, Y., Zhang, X., Chen, Y., 2015. Inhibition of porcine reproductive and respiratory syndrome virus by Cecropin D in vitro. Infect. Genet. Evol. 34, 7–16. Murtaugh, M.P., Stadejek, T., Abrahante, J.E., Lam, T.T.Y., Leung, F.C.C., 2010. The ever-expanding diversity of porcine reproductive and respiratory syndrome virus. Virus Res. 154, 18–30. Music, N., Gagnon, C.A., 2010. The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis. Anim. Health Res. Rev. 11, 135–163. Qiu, M., Chen, Y., Chu, Y., Song, S., Yang, N., Gao, J., Wu, Z., 2013. Zinc ionophores pyrithione inhibits herpes simplex virus replication through interfering with proteasome function and NF-kappaB activation. Antivir. Res. 100, 44–53. Rivara, S., Milazzo, F.M., Giannini, G., 2016. Heparanase: a rainbow pharmacological target associated to multiple pathologies including rare diseases. Future Med. Chem. 8, 647–680. Shi, M., Lam, T.T., Hon, C.C., Hui, R.K., Faaberg, K.S., Wennblom, T., Murtaugh, M.P., Stadejek, T., Leung, F.C., 2010. Molecular epidemiology of PRRSV: a phylogenetic perspective. Virus Res. 154, 7–17. Sun, Z., Chen, Z., Lawson, S.R., Fang, Y., 2010. The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions. J. Virol. 84, 7832–7846. te Velthuis, A.J., van den Worm, S.H., Sims, A.C., Baric, R.S., Snijder, E.J., van Hemert, M.J., 2010. Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture. PLoS Pathog. 6, e1001176. Yang, Y., Gorzelanny, C., Bauer, A.T., Halter, N., Komljenovic, D., Bauerle, T., Borsig, L., Roblek, M., Schneider, S.W., 2015. Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity. Oncogene 34, 5832–5842. Yun, S.I., Lee, Y.M., 2013. Overview: replication of porcine reproductive and respiratory syndrome virus. J. Microbiol. 51, 711–723. Zhang, L., Liu, J., Bai, J., Du, Y., Wang, X., Liu, X., Jiang, P., 2013. Poly(I:C) inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3. Antivir. Res. 99, 197–206. Zhang, Q., Huang, C., Yang, Q., Gao, L., Liu, H.C., Tang, J., Feng, W.H., 2016. MicroRNA30c modulates type I IFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1. J. Immunol. 196, 2272–2282. Zhao, C., Liu, S., Li, C., Yang, L., Zu, Y., 2014. In vitro evaluation of the antiviral activity of the synthetic epigallocatechin gallate analog-epigallocatechin gallate (EGCG) palmitate against porcine reproductive and respiratory syndrome virus. Viruses 6, 938–950.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Pyrithione inhibits porcine reproductive and respiratory syndrome virus replication through interfering with NF-kB and heparanase Chunhe Guo, Zhenbang Zhu, Xiaoying Wang, Yaosheng Chen, Xiaohong Liu * State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou Higher Education Mega Center, North Third Road, Guangzhou, Guangdong 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2016 Accepted 28 January 2017
Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccination strategies provide only limited protection against PRRSV infection. Consequently, there is a need to develop new antiviral strategies. Pyrithione (PT), a zinc ionophore, is used as an antibacterial and antifungal agent, and evidence has shown that PT inhibits the replication of various RNA viruses. However, there is no data regarding its effects against PRRSV infection until now. In this study, we showed that PT strongly inhibited PRRSV replication in Marc-145 cells. Similar inhibitory effects were also found in porcine alveolar macrophages, the major target cell type of PRRSV infection in pigs in vivo. PT also attenuated virus-induced apoptosis during the late phase of infection. In addition, we provided evidence that PT caused a rapid import of extracellular zinc ions into cells, and imported Zn2+ was responsible for its antiviral property. We investigated the molecular mechanisms of PT against PRRSV and found that PT inhibited NF-kB and heparanase, producing the increased heparan sulfate expression to block the release of virus and cytokines, thus decreasing viral replication. These findings suggest that PT has the potential to the development of prophylactic and therapeutic strategies against PRRSV infection. ß 2017 Elsevier B.V. All rights reserved.
Keywords: PRRSV Pyrithione Antiviral activity.
1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant porcine infectious diseases in the pig industry worldwide. It was first reported at the end of the 1980s in North America and Canada, and is commonly known as blue-eared pig disease (Zhao et al., 2014). The disease is characterized by abortion and poor reproductive performance in pregnant sows, respiratory distress in piglets and growing pigs, and high mortality in piglets (Music and Gagnon, 2010; Yun and Lee, 2013). PRRS virus (PRRSV), the etiological agent of the disease, belongs to the Arteriviridae family, together with equine arteritis virus, lactate dehydrogenaseelevating virus, and simian hemorrhagic fever virus. Like other arteriviruses, the viral genome is an enveloped, single, positivesense RNA strand of approximately 15 kb with a 50 -untranslated region (UTR), nine open reading frames (ORFs 1a, 1b, 2a, 2b, and
* Corresponding author. Tel.: +86 20 39332788; fax: +86 20 39332940. E-mail addresses: [email protected] (C. Guo), [email protected] (Z. Zhu), [email protected] (X. Wang), [email protected] (Y. Chen), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.vetmic.2017.01.033 0378-1135/ß 2017 Elsevier B.V. All rights reserved.
3–7) and a 30 -UTR (Fang and Snijder, 2010). According to its genetic diversity and geographic distribution, PRRSV can be broadly divided into two distinct genotypes with clear genetic and antigenic differences: the European type Lelystad strain (type 1) and the North American type VR-2332 strain (type 2), both of which share about 60% nucleotide sequence identity (Murtaugh et al., 2010; Shi et al., 2010). Pigs persistently infected with PRRSV develop viremia, delayed onset and low titer of neutralizing antibodies and reduced cellmediated immunity, they may shed virus for several months (Hu et al., 2012). Important features of PRRSV are its extreme genetic, antigenic, and immunobiological variability (Guo et al., 2014). Consequently, PRRSV remains the great challenge to swine industry, which highlights the need for novel and effective strategies to control PRRSV transmission (Guo et al., 2015a). Zinc ions are involved in many different cellular processes and have proven crucial for the proper folding and activity of various cellular enzymes and transcription factors. Maintaining the appropriate Zn2+ concentration is critical for cell survival since both increased and decreased Zn2+ levels can trigger apoptosis in a variety of cell types (te Velthuis et al., 2010). It is also an important
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cofactor for numerous viral proteins (te Velthuis et al., 2010). Previous studies have shown that polyprotein processing and protease activity are inhibited by Zn2+ in cells infected with human rhinovirus and coxsackievirus B3 (te Velthuis et al., 2010). Pyrithione (PT), a zinc ionophore, commonly acts as a centrosymmetric dimmer to increase the intracellular Zn2+ concentration via its oxygen and sulfur centers (Qiu et al., 2013). It is approved by Food and Drug Administration as worldwide, over-the-counter, topical antimicrobials for psoriasis and epidermal hyperplasia. PT is used as an antibacterial and antifungal agent, and evidence has shown that PT inhibits the replication of various RNA viruses, including influenza virus, respiratory syncytial virus and several picornaviruses (te Velthuis et al., 2010). In this study, we demonstrated that PT exhibited potent antiviral activity against PRRSV infection both in Marc-145 cells and pocine alveolar macrophage (PAMs). Mechanistic study showed that PT inhibited PRRSV replication via interfering with NF-kB and heparanase. 2. Materials and methods 2.1. Cells and viruses Porcine alveolar macrophages (PAMs) were obtained with lung lavage from the lungs of 3–8-week-old PRRSV-negative piglets (Guo et al., 2015b) and cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAA, Pasching, Austria), 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate at 37 8C in 5% CO2. All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Marc-145 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) containing 10% of FBS. Highly pathogenic PRRSV (HP-PRRSV) strain Li11 was propagated and titrated in PAMs or Marc-145 cells and used throughout the study. 2.2. Cell viability assay The viability of Marc-145 cells and PAMs after PT treatment was determined using the alamarBlue1 assay (Invitrogen, CA, USA) which is used to establish relative cytotoxicity of agents. Briefly, a series of concentrations of PT, diluted in DMEM or RPMI-1640, was applied to 60%–70% confluent Marc-145 cells or PAMs. After incubation for 48 h at 37 8C in a 5% CO2 atmosphere, 10 ml of alamarBlue1 was added and the cultures were incubated for another 3 h. Fluorescence intensity was detected at 570 nm excitation and 590 nm emission wavelengths and directly proportional to the number of living cells in culture. 2.3. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR)
After incubating for the indicated time, cells were rinsed with phosphate-buffered saline (PBS) three times and blocked with 1% Bovine serum albumin (BSA) in PBS for 30 min, then incubated with anti-PRRSV N protein mAb (SDOW17, Jeno Biotech Inc, Republic of Korea), anti-NF-kB p65 mAb (Cell Signal Technolgy (CST), MA, USA), anti-cleaved caspase-3 mAb (CST) or anti-heparan sulfate (HS) mAb (abcam, MA, USA) and followed by Alexa Fluor1 555-conjugated anti-mouse IgG secondary antibody for 2 h. After three washes in PBS, cell nuclei were stained with Hoechst dye 33258 (Sigma, MO, USA) and detected using fluorescence microscopy (Carl Zeiss, Jena, Germany). 2.5. Western blot analysis Marc-145 cells or PAMs cultured in six-well plates were harvested in lysis buffer (Beyotime, Jiangsu, China) containing a cocktail of protease inhibitors (Roche) and separated by 10% SDSPAGE, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After the membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris [pH 7.5], 150 mM NaCl, 0.5% Tween 20) for 2 h at 37 8C, they were rinsed and incubated with anti-PRRSV N protein mAb (SDOW17), antiheparanase mAb (abcam), anti-NF-kB p65 mAb (CST), antiglyceraldehyde phosphate dehydrogenase (GAPDH) mAb (CST), horseradish-peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (CST). Protein bands were visualized with ECL Plus chemiluminescence reagent (Pierce, Rockford, USA). 2.6. Virus titration assay In order to analyze the growth of PRRSV, the viral supernatants from cell cultures were collected at indicated time points after virus infection. Virus yields were measured by the 50% tissue culture infected dose (TCID50) assay using the Reed–Muench method. Briefly, cells were seeded in 96-well plates before virus infection. Viral supernatants were prepared by 10-fold serial dilution and 100 ml of dilutions were added per well in eight replicates. Virus titers were calculated 4–5 days post infection. 2.7. Apoptosis assay Apoptosis was evaluated by an Annexin V-FITC/Propidium Iodide (PI) assay (BD Biosciences Pharmingen, CA, USA) according to the manufacturer’s protocol. Marc-145 cells were mockinfected or infected with PRRSV at an multiplicity of infection (MOI) of 0.1 in the presence of PT for 36 h, then resuspended in 1 Binding Buffer after washed twice with cold PBS. Finally cells were stained with FITC Annexin V and PI for 15 min at RT in the dark and analyzed by flow cytometry (BD FACSCalibur, USA) within 1 h. 2.8. Antiviral assay
Total RNA was isolated from Marc-145 cells or PAMs using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions, and subjected to qRT-PCR analysis. cDNA was synthesized using reverse transcription kit (Promega, Madison, WI). SYBR Green (TaKaRa, Osaka, Japan) real-time PCR was performed using Light-Cycler 480 PCR system (Roche, Basel, Switzerland). Relative quantities of mRNA accumulation were D evaluated using the 2 Ct method. Primers will be made available upon request.
Marc-145 cells or PAMs were seeded in six-well plates and grown to 60%–70% confluence at 37 8C in 5% CO2, then inoculated with PRRSV at an MOI of 0.1 in the presence of different concentrations of PT. After incubation for 24 h, the supernatants were collected to titrate the viral yields, and the viral titers were determined as TCID50 (Delogu et al., 2011). The cells were harvested for IFA, qRT-PCR, and western blot analysis. 2.9. Viral release assay
2.4. Immunofluorescence assay (IFA) Cells were seeded onto coverslips, and then fixed with 4% paraformaldehyde and permeabilized at room temperature (RT).
Marc-145 cells were washed three times with PBS and then treated with PT at 37 8C for 4 h after challenged with PRRSV (MOI = 0.1) at 37 8C for 24 h. The cell culture supernatants were
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collected to titrate the viral yields and to quantify viral particles using TCID50 and qRT-PCR methods. 2.10. Cell fractionation Marc-145 cells were fractionated using a Nuclear/Cytosol Fractionation Kit (BioVision, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were washed twice with icecold PBS, and transferred to 1.5 ml Eppendorf tubes, and then centrifuged at 600 g for 5 min at 4 8C. The pellet was resuspended in cold Cytosol Extraction Buffer A and B. After centrifugation at maximal speed for 5 min, the cytoplasmic extract fraction was transferred to a clean pre-chilled tube. For the nuclear extract, the pellet was resuspended in cold Nuclear Extraction Buffer A and centrifuged at full speed for 10 min. The extracted nuclear and cytoplasmic protein fractions were prepared for western blot analysis. 2.11. Statistical analysis All experiments were performed with at least three independent replicates. All data were presented as means standard errors (SE). Statistics analysis was performed by SPSS 16.0 using Student’s ttest and one-way ANOVA. Differences with P values <0.05 were considered significant.
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3. Results 3.1. PT strongly inhibits PRRSV infection and replication in Marc-145 cells To evaluate the in vitro antiviral activity of PT against PRRSV infection, cytotoxicity assay was essential for the initial phase of antiviral drug development. We first investigated its cytotoxicity on Marc-145 cells using alamarBlue1. As shown in Fig. 1A, no significant cytotoxicity was observed up to 80 mg/ml PT. Therefore, we performed all subsequent experiments with PT at a concentration no higher than 80 mg/ml in Marc-145 cells. Next cytopathic effects (CPE) observation, IFA and virus titers assays were performed to determine the effect of PT on PRRSV infection. PT (15 mg/ml) notably abrogated the PRRSV-induced CPE at 72 h post infection (hpi) in Marc-145 cells infected with PRRSV at an MOI of 0.1 (Fig. 1B). The specificity of CPE was further confirmed by IFA with anti-PRRSV N protein mAb at 24 hpi. Upon PT treatment, N-specific staining was notably reduced in a dosedependent manner in Marc-145 cells compared to the control without PT treatment (Fig. 1C), indicating that infection and the spread of the virus to the neighboring cells are blocked by PT. Consistent with these findings, the production of viral progeny was significantly reduced at 24 hpi by PT, in a dose-dependent manner (Fig. 1D).
Fig. 1. Antiviral activity of PT against PRRSV infection in Marc-145 cells. (A) Potential cytotoxicity of PT against Marc-145 cells was detected with alamarBlue1. A series of concentrations of PT was applied to 60%–70% confluent cells for 48 h, and then 10 ml of alamarBlue1 was added. (B) Cells were mock infected or infected with PRRSV at an multiplicity of infection (MOI) of 0.1 in the presence or absence of PT (15 mg/ml). Virus-produced cytopathic effects (CPE) were observed by bright-field microscopy at 72 h post infection (hpi). (C) Cells were fixed with 4% paraformaldehyde in the presence or absence of different concentrations of PT at 24 hpi, then analyzed by IFA using antiPRRSV N protein mAb (red). Bar, 200 mm. (D) Virus titers in virus-infected cells (MOI = 0.1) were determined followed by treatment with or without PT at 24 hpi. (E and F) Cells were inoculated with PRRSV (MOI = 0.1) in the presence or absence of different concentrations of PT for 24 h, the transcript (E) and protein (F) levels of PRRSV N gene were determined. Expression of GAPDH was shown as a loading control. (G and H) To evaluate whether PT can inhibit the level of N protein both in the nuclear and cytoplasmic fractions of PRRSV-infected cells. The amount of N protein both in the cytoplasm (G) and nucleus (H) in cells treated with or without PT was determined at 24 hpi. GAPDH and Histone H3 were used as cytoplasmic and nuclear controls, respectively. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
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Since viral RNA transcription and protein translation are functionally connected in PRRSV replication (Lee and Lee, 2010), we attempted to assess the transcript and protein levels of PRRSV N gene in the presence or absence of PT at 24 hpi in Marc-145 cells. As shown in Fig. 1E, the cells treated with PT showed a significant reduction in the transcript level of PRRSV N gene in a dosedependent manner compared to those without PT treatment. Consistently, the translation level of the viral N protein was decreased significantly by PT in comparison to the control (Fig. 1F). Since the N protein of PRRSV is found not only in the cytoplasm but also in the nucleus of virus-infected cells (Lee and Lee, 2016), we attempted to evaluate whether PT can inhibit the level of N protein both in the nuclear and cytoplasmic fractions of PRRSV-infected cells. As shown in Fig. 1G and H, the amount of N protein both in the nucleus and cytoplasm in PRRSV-infected cells was notably diminished in the presence of PT at 24 hpi. Taken together, these data indicate that PT exerts an inhibitory effect on PRRSV infection in Marc-145 cells. 3.2. PT inhibits PRRSV replication in PAMs Since PT effectively inhibited PRRSV infection and replication in Marc-145 cells, we investigated whether PT also inhibits PRRSV replication in PAMs, the major target cell type of PRRSV infection in pigs in vivo. We initially performed cell viability assay on PAMs according to the methods described in ‘‘Materials and methods’’. PT was noncytotoxic up to 60 mg/ml after incubation for 48 h (Fig. 2A). Next we evaluated its effect on PRRSV replication in PAMs. Consistent with the findings obtained with Marc-145 cells, PT (15 mg/ml) caused significant reductions in the viral N protein at the transcript and protein levels and in the viral titers at 24 hpi,
Fig. 2. PT inhibits PRRSV replication in PAMs. (A) Cytotoxic activity of PT against PAMs was measured using the alamarBlue1. PAMs were incubated with various concentrations of PT for 48 h. (B–D) PAMs were infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml) for 24 h. The viral titers in the supernatants (B), viral RNA transcription (C) and protein (D) levels were analyzed. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
Fig. 3. PT blocks PRRSV-induced apoptosis during the late phase of infection. (A) Marc-145 cells were mock-infected or infected with PRRSV at an MOI of 0.1 in the presence or absence of PT (15 mg/ml). After incubation for 36 h, cells were detected by IFA with anti-cleaved caspase-3 mAb (red). Bar, 200 mm. (B) Cells were mock-infected or infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml) for 36 h, then resuspended in 1 Binding Buffer after washed twice with cold PBS. Finally cells were stained with FITC Annexin V and PI for 15 min and analyzed by flow cytometry (BD FACSCalibur, USA) within 1 h. Q1, necrotic or another cell population that was FITC Annexin V-negative and PI-positive; Q2, end stage apoptotic or a dead cell population that was FITC Annexin V- and PI-positive; Q3, an early apoptotic cell population that was FITC Annexin V positive and PI-negative; Q4, a viable cell population that was not undergoing apoptosis and was both FITC Annexin V- and PI-negative.
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compared with those of cells without PT treatment (Fig. 2B–D). Taken together, PT inhibits PRRSV replication in PAMs as well. 3.3. PT blocks PRRSV-induced apoptosis during the late phase of infection Previous studies have shown that PRRSV stimulates antiapoptotic pathways in Marc-145 cells and PAMs early in infection and that PRRSV-infected cells die from apoptosis late in infection (Liu et al., 2015). To investigate whether PT can block virusinduced apoptosis during the late phase of infection, IFA with anticleaved caspase-3 mAb was performed. As shown in Fig. 3A, upon PT treatment (15 mg/ml), cleaved caspase-3-specific staining (red) was notably reduced in Marc-145 cells at 48 hpi compared to the control without PT treatment. Further quantification of apoptotic cells was performed with flow cytometry. The results showed that the apoptosis of PRRSV-infected cells were significantly decreased after treatment with PT at 48 hpi. Together, these data demonstrate that PT blocks PRRSV-induced apoptosis during the late phase of infection, which might contribute to its inhibition of PRRSV infection. 3.4. PT leads to a rapid import of extracellular zinc ions into cells To directly visualize the ability of PT to increase the intracellular pool of labile Zn2+, fluorescence microscopy with specific zinc indicators was employed. FluoZinTM-3 (Invitrogen, CA, USA) is suitable for detection of Zn2+ concentrations in the 1–100 nM range and it has shown to be a Zn2+-sensitive and Zn2+-specific fluorescent probe that has no specific cellular localization (Krenn et al., 2009). As shown in Fig. 4A and B, in cells loaded with
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FluoZinTM-3, intracellular fluorescence was minimal due to a very low level of free Zn2+. After the application of the zinc ionophore PT in cells, the fluorescence was significantly increased in dose- and time-dependent manners (Fig. 4A and B), indicating that the intracellular labile ‘‘chelatable’’ zinc level is elevated. 3.5. Zn2+ is responsible for the antiviral property of PT Since Zn2+ plays a vital role in the bioactivities of PT (Qiu et al., 2013), we investigated the importance of Zn2+ in PT inhibition of PRRSV replication. The specific role of Zn2+ was illustrated by the treatment of metal ion chelator EDTA-Mg (Sigma) and N,N,N0 ,N0 tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, Sigma). In the presence of EDTA-Mg (10 mM), PT (15 mg/ml) inhibition of the transcript and protein levels of viral N protein was partially abrogated since Zn2+ in the culture medium competes for binding EDTA-Mg to form higher affinity EDTA-Zn, thus depleting the free Zn2+ (Fig. 4C and D). Consistently, TPEN (3 mg/ml) was capable of mitigating the antiviral property of PT against PRRSV replication (Fig. 4E). These data indicates that the availability of Zn2+ is a prerequisite for the anti-PRRSV activity of PT. 3.6. PT inhibition of NF-kB activation might contribute to its antiPRRSV activity It is well known that PRRSV infection activates NF-kB pathway, an essential step for effective viral gene expression and replication (Zhang et al., 2016). To investigate whether PT can inhibit PRRSVinduced NF-kB activation in Marc-145 cells, confocal microscopy was performed. As shown in Fig. 5A, in PRRSV-infected cells, nuclear accumulation of endogenous p65 (red) was observed,
Fig. 4. PT increases the intracellular pool of labile Zn2+ which is responsible for its antiviral activity. (A and B) Cells were loaded with FluoZinTM-3 acetoxymethyl (AM) ester (Invitrogen, CA, USA) in PBS for 15 min, and incubated for a further 30 min in growth medium to allow complete de-esterification of intracellular AM esters. PT was then added to the medium for the indicated concentrations (A) and times (B). Fluorescence was monitored in a live-cell microscope system (Carl Zeiss, Jena, Germany) using fluorescein isothiocyanate settings for FluoZinTM-3. Bar, 200 mm. (C and D) Cells were infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml) and EDTA-Mg (10 mM) for 24 h, the transcript (C) and protein (D) levels of PRRSV N gene were determined by qRT-PCR and western blot analysis. (E) The transcript level of PRRSV N gene in infected cells in the presence or absence of PT (15 mg/ml) and TPEN (3 mg/ml) was analyzed at 24 hpi. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
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Fig. 5. PT inhibition of NF-kB activation might contribute to its anti-PRRSV activity. (A) Marc-145 cells were mock-infected or infected with PRRSV at an MOI of 0.1 in the presence or absence of PT (15 mg/ml) for 24 h, and then fixed with 4% paraformaldehyde and analyzed by IFA using anti-NF-kB p65 mAb (CST, MA, USA) (red). Bar, 25 mm. (B) To further confirm that PT can inhibit PRRSV-induced NF-kB activation, a cell fractionation assay in cells treated with or without PT (15 mg/ml) was performed at 24 hpi. GAPDH and Histone H3 were used as cytoplasmic and nuclear controls, respectively. (C–E) Virus-infected or uninfected PAMs were cultured in the presence of PT (15 mg/ml) for 18 h. The expression of IFN-a (C), IFN-b (D) and TNF-a (E) was analyzed by qRT-PCR. (F and G) Marc-145 cells were mock-infected or infected with PRRSV (MOI = 0.1) in the presence of PT (15 mg/ml) for 18 h. qRT-PCR was conducted to evaluate the expression of IL-6 (F) and TNF-a (G). Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
indicating that NF-kB is activated as expected. However, in virusinfected cells treated with PT (15 mg/ml), p65 mainly existed in cytoplasm, similar to the results of uninfected mock-treated cells (top panel). Consistent with above data, we performed a cell fractionation assay and found that PT (15 mg/ml) attenuated PRRSV-mediated p65 nuclear translocation (Fig. 5B), further confirming that PT inhibits PRRSV-induced NF-kB activation. NF-kB is an important transcription factor for cytokines production. PRRSV infection activates NF-kB and subsequent production of inflammatory cytokines. Whether PT can inhibit PRRSV-induced upregulation of cytokines is also investigated. PT (15 mg/ml) strongly inhibited the expression of IFN-a, IFN-b and TNF-a induced by PRRSV in PAMs (Fig. 5C–E). Consistently, the upregulation of IL-6 and TNF-a expression was significantly reduced by PT in PRRSV-infected Marc-145 cells (Fig. 5F and G). These results demonstrate that PT can interfere with the
production of NF-kB-mediated cytokines caused by PRRSV infection. 3.7. PT inhibits PRRSV-induced heparanase Previous studies have shown that heparanase is implicated in the regulation of various physiological and pathological processes (Rivara et al., 2016). Upon herpes simplex virus-1 (HSV-1) infection, heparanase is upregulated through NF-kB pathway and translocated to the cell surface to facilitate viral release (Hadigal et al., 2015). Initially, we investigated whether PRRSV can induce heparanase expression in Marc-145 cells. Western blot and qRT-PCR analysis revealed that expression of heparanase was significantly greater at 36 hpi than in uninfected cells (Fig. 6A and B). Next we evaluated the effect of PT on heparanase induced by PRRSV in Marc-145 cells. As shown in Fig. 6C and D, in the presence
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of PT (15 mg/ml), the transcript and protein levels of heparanase were significantly reduced at 36 hpi. Collectively, these data suggest that PT is able to inhibit PRRSV-induced heparanase. 3.8. PT inhibition of HS loss from cell surface after infection might contribute to its capacity of blocking viral release
Fig. 6. PT inhibits PRRSV-induced heparanase. (A and B) Cells were mock-infected or infected with PRRSV at an MOI of 0.1 for 36 h, and then harvested for the transcript (A) and protein (B) levels of heparanase by qRT-PCR and western blot analysis. (C and D) Virus-infected cells were cultured in the presence or absence of PT (15 mg/ ml) for 36 h. Heparanase transcription (C) and protein (D) levels were analyzed. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
HS is a ubiquitously expressed glycosaminoglycan that is used as a docking site for initial attachment by many viruses such as dengue virus, foot-and-mouth disease virus and PRRSV (Hadigal et al., 2015). However, during a productive infection, the HS moieties on parent cells can trap newly exiting viral progenies and block viral release (Hadigal et al., 2015). To determine whether PT can inhibit the loss of HS from cell surface after PRRSV infection in Marc-145 cells, qRT-PCR and confocal microscopy with anti-HS mAb assays were performed. As shown in Fig. 7A and B, a dramatic decrease in HS was seen during later stage of infection (36 hpi) in Marc-145 cells compared with that in uninfected mock-treated cells. However, in the presence of PT (15 mg/ml), HS from the surface of PRRSV-infected cells was significantly increased at 36 hpi, indicating that PT can inhibit HS degradation during later stage of PRRSV infection. Since HS is able to block viral release during a productive infection, we investigated whether PT can inhibit PRRSV release in Marc-145 cells. Our results showed that significantly fewer infectious viral particles were released into the supernatant by cells treated with PT (15 mg/ml) for 4 h than by untreated cells at 36 hpi (Fig. 7C). The viral titers in the supernatants showed a similar pattern after treatment with PT (15 mg/ml) for 4 h (Fig. 7D). Taken together, these data indicate that PT inhibits HS degradation
Fig. 7. PT inhibition of HS degradation might contribute to its capacity of blocking viral release. (A) Cells were fixed with 4% paraformaldehyde in the presence or absence of PT (15 mg/ml) at 36 hpi, then analyzed by IFA using anti-HS mAb (red). Bar, 25 mm. (B) Cells were mock-infected or infected with PRRSV (MOI = 0.1) in the presence or absence of PT (15 mg/ml). After incubation for 36 h, cells were collected to determine RNA transcription of HS. (C and D) Cells were infected with PRRSV at an MOI of 0.1 for 36 h, and then treated with PT (15 mg/ml) for 4 h. The supernatants were harvested to measure extracellular viral particles (C) and viral titers (D) using qRT-PCR and TCID50, respectively. Data are representative of the results of three independent experiments (means SE). Significant differences compared with control group are denoted by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).
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Fig. 8. Schematic model of anti-PRRSV activity of PT. During the productive period of infection, PRRSV activates NF-kB to promote HPSE mRNA expression, then the cytoplastic heparanase are translocated to the cell surface and exert the enzymatic activity to process the ECM, resulting in the release of virus and cytokines attached to fragments of ECM-resident HS. As an inhibitor of NF-kB and HPSE, PT can inhibit this effect.
induced by PRRSV during later stage of infection, which might contribute to its inhibition of viral release. Heparanase is the only known mammalian endoglycosidase that cleaves HS, facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines in a number of physiological and pathological settings (Yang et al., 2015). Previous studies have shown that HSV-infected cells upregulate heparanase through NF-kB, which is then translocated to the cell surface, producing the decreased HS expression to allow viral exit (Hadigal et al., 2015). Based in our results, the inhibitory mechanisms of PT may be illuminated in the model (Fig. 8). During the productive period of infection, PRRSV activates NF-kB to promote HPSE mRNA expression, then the cytoplastic heparanase are released to the extracellular space and exert the enzymatic activity to process the ECM, resulting in release of virus and cytokines attached to fragments of ECMresident HS. As an inhibitor of NF-kB and heparanase, PT can inhibit this effect. 4. Discussion PRRSV first emerged in the late 1980s in North America, and subsequently in Europe, causing huge economic losses. It has since spread across the globe, inducing characteristic severe reproductive failure in female pigs and respiratory tract illness in piglets. PRRSV primarily infects PAMs and is characterized by the high rate of mutation and recombination (Deaton et al., 2014). Moreover, it causes the delayed appearance and low titer of neutralizing antibodies and initiates a comprehensive campaign against the innate immune response (Fang et al., 2012; Sun et al., 2010). Therefore, there is an urgent need for developing new antiviral strategies against PRRSV infection. PT, known to be a zinc ionophore that leads to a rapid increase in intracellular zinc levels, is effective against several pathogens such as human rhinovirus, coxsackievirus, mengovirus and HSV (Qiu et al., 2013).
In the current study, we showed that PT exhibited potent antiviral activity against PRRSV infection and replication in vitro at noncytotoxic concentrations (Figs. 1 and 2). Mechanistic study showed that PT inhibited the expression of heparanase via interfering with NF-kB activation, and then blocked the loss of HS from the cell surface, thus inhibited the release of virus and cytokines (Fig. 8). We provided evidence that the basis of the antiviral activity was dependent upon the availability of zinc ions as this inhibitory activity could be blocked by the addition of Zn2+ chelators, such as EDTA-Mg and TPEN (Fig. 4). The import of extracellular zinc ions is a key feature of the antiviral property of PT, which is consistent with previous reports (Krenn et al., 2009). The PRRSV N protein, the most abundant viral protein expressed in infected cells, blocks host protein synthesis due to the high concentration in the nucleolus (Jourdan et al., 2012). Moreover, the N protein may impact transcriptional regulation in infected cells owing to its interacting with transcriptional regulators (Dokland, 2010). In this study, our data demonstrated that PT effectively decreased the transcription and translation levels of the viral N protein (Figs. 1 and 2). Moreover, PT exhibited robust anti-PRRSV activity via multiple pathways including inhibition of virus progeny production, virus-induced apoptosis during the late phase of infection, and viral particle release (Figs. 1, 3 and 7). Since Marc-145 cells are not of porcine origin but are monkey cells (Zhang et al., 2013), we also examined the antiviral activity of PT in PAMs, which are known to be the primary host cell target for PRRSV infection in vivo. Consistent with the data obtained with Marc-145 cells, PT strongly inhibited virus replication in PAMs (Fig. 2), indicating that it might be an effective inhibitor of PRRSV infection in vivo. HS is a key component of the ECM and is used by many viruses for initial attachment to target cells. As the only known enzyme capable to degrade HS, heparanase mediates the release of HSbound cytokines, growth factors and viruses (Yang et al., 2015). Previous studies have demonstrated that HS expression is
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increased during the initial stages of HSV-1 infection to enhance viral attachment, while a dramatic decrease in HS is seen during later stages of infection. The results indicate that heparanase is upregulated in the loss of HS late during HSV-1 infection. In this study, we showed that expression of heparanase was elevated and a dramatic decrease in HS was seen during later stage of PRRSV infection (36 hpi, Figs. 6 and 7), which is consistent with above research. This identifies a novel role for heparanase and HS in the regulation of PRRSV infection. However, further research should be undertaken to investigate the expression of HS and heparanase during the initial stages of PRRSV infection and whether viral attachment and detachment modes are regulated by the host enzyme heparanase. In physiological conditions, heparanase is expressed at high levels only in few tissues while in specific pathological conditions, it is upregulated and highly affects multiple biological processes, indicating that it might be recognized as a negative prognostic marker (Rivara et al., 2016). Heparanase is involved in many different pathological scenarios such as inflammatory diseases, angiogenesis, cancer and metastasis (Rivara et al., 2016). Therefore, heparanase has attracted considerable attention as a promising target for innovative pharmacological applications. PI-88, a potent heparanase inhibitor that is currently in human clinical trials for cancer treatment, can reduce viral release and control the host inflammatory response (Rivara et al., 2016). In this study, we demonstrated that heparanase was upregulated in PRRSV-infected cells while in the presence of PT, it was significantly decreased at 36 hpi (Fig. 6), indicating that PT inhibits viral replication via interfering with heparanase which might be a unique therapeutic target against PRRSV. In conclusion, the results presented herein demonstrate that PT exerts a potent inhibitory effect on PRRSV through interfering with NF-kB and heparanase, indicating that PT has the potential to the development of prophylactic and therapeutic strategies against PRRSV infection. Further research is required to evaluate the potential in vivo antiviral activity of PT in animal models. Competing interests The authors declare that they have no competing interests. Acknowledgements This work was supported by National Natural Science Foundation of China (31601917), Natural Science Foundation of Guangdong Province (2014A030312011) and Guangdong Sailing Program (2014YT02H042). References Deaton, M.K., Spear, A., Faaberg, K.S., Pegan, S.D., 2014. The vOTU domain of highlypathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference. Virology 454-455, 247–253. Delogu, I., Pastorino, B., Baronti, C., Nougairede, A., Bonnet, E., de Lamballerie, X., 2011. In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant. Antivir. Res. 90, 99–107. Dokland, T., 2010. The structural biology of PRRSV. Virus Res. 154, 86–97. Fang, Y., Snijder, E.J., 2010. The PRRSV replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins. Virus Res. 154, 61–76.
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