Research in Veterinary Science 96 (2014) 323–327

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Antiviral activity and underlying molecular mechanisms of Matrine against porcine reproductive and respiratory syndrome virus in vitro Na Sun a, Zhi-Wei Wang a, Cai-Hong Wu b, E. Li a, Jun-Ping He a, Shao-Yu Wang c, Yuan-Liang Hu d, Hai-Min Lei e, Hong-Quan Li a,⇑ a

College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801, PR China Jiangsu Animal Husbandry & Veterinary College, Taizhou, Jiangsu 225300, PR China University of Western Sydney, School of Medicine, Locked Bag 1797, Penrith 2751, NSW, Australia d Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China e School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, PR China b c

a r t i c l e

i n f o

Article history: Received 12 September 2013 Accepted 15 December 2013

Keywords: Matrine Antiviral PRRSV N protein Apoptosis

a b s t r a c t Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is an acute infectious disease. The prevalence of PRRS has made swine industry suffered huge financial losses. Matrine, a natural compound, has been demonstrated to possess antiPRRSV activity in Marc-145 cells. However, the underlying molecular mechanisms were still unknown. The main objective of our study was to discuss the effect of Matrine on PRRSV N protein expression and PRRSV induced apoptosis. Indirect immunofluorescence assay (IFA) and Western blot were used to assess the effect of Matrine on N protein expression. Apoptosis was analyzed by fluorescence staining. In addition, the effect of Matrine on caspase-3 activation was investigated by Western blot. Indirect immunofluorescence assay and Western blot analysis demonstrated that Matrine could inhibit N protein expression in Marc-145 cells. And Matrine was found to be able to impair PRRSV-induced apoptosis by inhibiting caspase-3 activation. Ó 2014 Published by Elsevier Ltd.

1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is a devastating swine disease worldwide. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is a singlestranded RNA virus in the Arteriviridae family, order Nidovirales. Its genome with the size of approximately 15 kb encodes at least eight open reading frames (ORFs). ORF7 encodes the nucleocapsid (N) protein which is the most abundant and immunogenic viral protein among all proteins in PRRSV (Dokland, 2010). Porcine reproductive and respiratory syndrome was first discovered in China in 1996 and a severe outbreak of this syndrome occurred in 2006 which affected more than two million pigs across more than ten provinces in China (Tian et al., 2007). The currently available measure for controlling and preventing the syndrome mainly depends on vaccination. The vaccines suffer from problems in either controlling complicated outbreaks or genetic variants of the virus. Moreover, PRRS can suppress immune system. Therefore, finding safe, effective and inexpensive ways to control PRRSV infection is impetus.

⇑ Corresponding author. Tel./fax: +86 354 6288409. E-mail address: [email protected] (H.-Q. Li). 0034-5288/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.rvsc.2013.12.009

Traditional Chinese medicines, science antiquity to date, have been widely used to treat infectious diseases. Matrine, a major quinolizidine alkaloid purified from the dried roots of Sophora flavescens Ait (Chinese Pharmacopoeia 2005), has been found to display multi-pharmacological effects including antivirus, antiinflammation, and immunity-regulation (Azzam et al., 2007; Cao et al., 2011; Jiang et al., 2007; Ma et al., 2008). Our previous research data showed that Matrine could inhibit PRRSV infection on Marc-145 cells and the mode of antiviral action of Matrine on PRRSV infection may be able to directly inactivate PRRSV and interfere with its replication within cells (Zhao et al., 2013). All these results were encouraging to further explore the exact molecular mechanisms on anti-PRRSV of Matrine. In this study, effects of Matrine on PRRSV N protein expression and PRRSV induced apoptosis were evaluated to further clarify the molecular mechanism of Matrine against PRRSV in vitro. 2. Materials and methods 2.1. Test compounds Matrine and Ribavirin were purchased from National Institutes For Food and Drug Control (Beijing, PR China). The batch numbers were respectively 110805-200508 and 140629. The purity of

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Matrine was greater than 99%. These compounds were dissolved and diluted in DMEM (Dulbecco’s Modified Eagle Medium, Sigma, USA) with 2% FCS (fetal calf serum, Hyclone, USA). Ribavirin was used as a positive control (Kim and Lee, 2013). In the previous study, cytotoxicity and anti-PRRSV activity of Matrine were measured with MTT test and the analyses of cytopathogenic effect (CPE) in Marc-145 cells. These results showed that the maximum non-cytotoxic concentration (MNTC) of Matrine and Ribavirin were 0.75 and 0.125 mg/ml, respectively. And the maximum inhibition ratio of Matrine and Ribavirin were 93.6 and 61.4, respectively. When the concentration of Matrine was lower than 0.1875 mg/ml, the inhibition ratio was lower than 20% (Zhao et al., 2013). In order to display dose-dependent and ensure noncytotoxicity, the first three concentrations of Matrine (0.75, 0.375 and 0.1875 mg/ml) were used to evaluate the effect on PRRSV N protein expression in the following experiment. 2.2. Cells and virus Marc-145 cells, a subclone of the African green monkey kidney epithelial cell line, obtained from China Institute of Veterinary Drug Control (Beijing, PR China), were grown in DMEM supplemented with 10% FCS, 100 IU/ml penicillin G and 100 lg/ml streptomycin at 37 °C in a humidified cabinet containing 5% CO2 atmosphere. PRRSV vaccine (JXAI-R, No. 1012001, Guangdong Dahuanong Animal Health Products Co., Ltd., PR China) was propagated in Marc-145 cells. Virus titers, calculated by the method of Reed and Muench (Reed and Muench, 1938), was 107.5 TCID50/ml. Virus liquids were stored at 80 °C till use. 2.3. Indirect immunofluorescence assay (IFA) Procedures for IFA were carried out as previously reported (Sun et al., 2012) with some modifications. In one procedure, the confluent monolayer of Marc-145 cells in 384-well culture plates were infected with 20 ll of each of three different concentrations of Matrine and a constant amount of 100 TCID50 PRRSV. In the other procedure, cells were pre-infected with 20 ll PRRSV for 2 h at 37 °C, virus medium was then removed and washed thrice in PBS, and finally each of three different concentrations of Matrine were added. All these plates were incubated at 37 °C in a 5% CO2 humidified for 48 h. All cells were then fixed with cold mixtures of acetone and methanol (1:1) for 30 min at 20 °C and washed thrice in PBS. The infected cells were incubated with monoclonal antibodies against N protein (1:50) for 2 h at 37 °C and washed thrice in PBS. Afterwards fixed cells were incubated for 1 h at 37 °C in FITC-conjugated goat anti-mouse IgG (Bioss, Beijing, PR China) at 1:30 dilution and washed thrice in PBS. The infected cells treated with antibodies were immediately visualized with fluorescence microscope. Cells were also stained with DAPI (40 ,6-diamidino-2phenylindole, 1:20, KeyGen, Nanjing, PR China). 2.4. N protein determination by Western blot In parallel, N protein expression in the cells was determined using Western blot. Above-mentioned cell samples (described in Section 2.3) were prepared. The total protein was extracted using a total protein extraction kit (Applygen, PR China) according to the manufacturer’s instructions. Protein concentration was estimated by NanoDrop spectrophotometer (ND-1000, USA). After heat denaturation at 95 °C for 5 min. 20 lg of protein were loaded on sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) with a 4% stacking gel and a 12% gradient gel. At the end of the electrophoresis, proteins were transferred to a PVDF membrane and then blocked with 5% skim milk in TBST with gentle shaking at room

temperature for 2 h. The membrane was incubated with anti-N protein antibody (diluted 1:1000) at room temperature for 2 h. After washing the membrane with TBST thrice, membrane strips were incubated with a 1:5000 dilution of HRP (horseradish peroxidase) conjugated anti-mouse IgG at 37 °C for 1 h with gentle shaking and then washed with TBST thrice again. Detection was performed with enhanced chemiluminescence (eECL kit, Cowin Biotech Co., Ltd., China) and recorded on an X-ray film (Kodak). b-Actin was determined as a loading control to normalize samples. 2.5. Cell apoptosis determination with fluorescence microcopy Cell apoptosis was analyzed using fluorescence microscope as previously described with some modifications (Sui et al., 2010). Briefly, the confluent monolayer of Marc-145 cells in 384-well plates was prepared, and then Matrine and PRRSV were added (the finally concentration of Matrine was 0.75 mg/ml, and virus concentration was 100 TCID50). Cells control and PRRSV control were set up simultaneously. After 24, 48 and 60 h incubation at 37 °C in a 5% CO2 humidified atmosphere, cells were separately treated according to the protocol of the AnnexinV-EGFP Kit (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, PR China) and then analyzed by fluorescence microscope. 2.6. Detection of caspase-3 using Western blot Cell samples were prepared at 48 h post-infection as described in the above section. The protein samples (50 lg) were separated by SDS–PAGE with a 4% stacking gel and a 15% gradient gel. The proteins in SDS–PAGE were transferred onto PVDF membranes and blocked with TBST containing 5% skim milk at room temperature for 2 h. Then the membranes were incubated with rabbit anticaspase-3 (diluted 1:1000, Cell Signaling Technology, #9662) and mouse anti-b-actin (diluted 1:1000, CWBIO, #CW0096) at 4 °C overnight with gentle shaking. After washing the membrane with TBST thrice, membrane strips were incubated with different secondary antibodies matching to their respective primary antibodies at 1:5000 dilutions at 37 °C for 1 h with gentle shaking and then washed with TBST thrice again. Protein detection was performed with enhanced chemiluminescence (eECL kit, Cowin Biotech Co., Ltd., PR China) and recorded on an X-ray film (Kodak). 3. Results To determine the inhibitory effect of Matrine on N protein, Marc-145 cells were simultaneously infected with PRRSV and Matrine or pre-incubated with PRRSV followed by Matrine. The expression of the N protein of these treated cell samples were then analyzed by IFA and Western blot. The data showed that Matrine inhibited N protein expression in a dose dependent manner. As shown in Fig. 1, with the lower concentration of Matrine, the more N protein (green florescence) was detected. From Fig. 1D and G, or 1E and H, even if at the same concentration of Matrine, the N protein expression levels were different according to the modes of adding the compound. Obviously, the expression of N protein was less in the mode of adding PRRSV and Matrine simultaneously onto cells. And the results of Western blot were also consistent with the data from the IFA, demonstrating that Matrine could both inactive PRRSV directly and interfere with PRRSV replication. In addition, from Fig. 1E and G, we observed a characteristic pattern of small foci of positive cells, suggesting the drug may inhibit PRRSV dissemination. The cell apoptosis was analyzed with Annexin V-EGFP and PI double staining. The results showed that PRRSV induced early and late apoptosis (Fig. 2). As indicated in Fig. 3A, PRRSV-infected

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Fig. 1. Effect of Matrine on PRRSV N protein expression in 384-well plates at 48 h post-infection. (A) Cell control; (B) PRRSV control; (C) Ribavirin control; (D–F) Marc-145 cells were simultaneously infected with PRRSV and different concentration of Matrine (D: 0.75 mg/ml; E: 0.375 mg/ml F: 0.1875 mg/ml); (G–I) different concentrations of Matrine were added to cells after PRRSV infection, (G: 0.75 mg/ml; H: 0.375 mg/ml; I: 0.1875 mg/ml). N protein was stained with the FITC (green). Cell nuclei were stained with DAPI (blue). The expression of N protein using Western blot were shown in Panel I and Panel II. Panel I: Cells were simultaneously infected with PRRSV and Matrine with three different concentrations. Panel II: different concentrations of Matrine were added to cells after PRRSV infection. (lane 1: cell control; lane 2: PRRSV control; lane 3: Ribavirin control; lane 4: 0.75 mg/ml; lane 5: 0.375 mg/ml; lane 6: 0.1875 mg/ml of Matrine). Magnification, 40. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

cells show little staining which was same as to the cells in cell control at 24 h post infection. But the number of apoptosis cells was significantly increased at 48 and 60 h. However, the number of apoptosis cells of PRRSV-infected cells treated with Matrine was significantly fewer than the PRRSV-infected cells, indicating the inhibiting action to apoptosis (Fig. 3A and B). To further confirm the inhibiting effect of Matrine on cell apoptosis, caspase-3 activity was measured. As shown in Fig. 4, caspase-3 was not activated until 48 h of PRRSV infection, and Matrine could prevent caspase-3 activation at 48 h post-infection, which was consistent with those observed in Fig. 3. 4. Discussion Fig. 2. PRRSV could induce both early and late apoptosis. Cell apoptosis was measured by fluorescence staining of AnnexinV-EGFP Kit after PRRSV infection. In early apoptotic cells, only green fluorescence was found and they are around cell membrane; while in late apoptotic cells, green fluorescence was found around cell membrane and red fluorescence in nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Our previous research has been shown Matrine could inhibit PRRSV infection in Marc-145 cells by using CPE and MTT assay (Zhao et al., 2013). In this study, we further confirmed Matrine possessed the ability of inhibiting PRRSV N protein expression and

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A

number of apoptotic cells

B 1500

Cell control

1200

Cell+Matrine

PRRSV control Cell+PRRSV+Matrine

900 600 300 0

48h

60h

hour post-infection(h.p.i.) Fig. 3. Effect of Matrine on PRRSV-induced cell apoptosis. (A) Effect of Matrine on PRRSV induced apoptosis was measured by fluorescence staining after 24, 48 and 60 h post-infection. Six independent wells were performed for each group. After 48 h and 60 h co-incubation with PRRSV and Matrine, the PRRSV-induced cell apoptosis was attenuated comparing with PRRSV control. Magnification, 40. (B) The corresponding number of apoptotic cells in different treatment group.

Fig. 4. Effect of Matrine on caspase-3 activation was measured by Western blot analysis. Cleaved caspase-3 expression was detected in Marc-145 cells infected with PRRSV at 48 h and undetectable in Matrine treated group. Matrine prevented such caspase-3 activation after 48 h post-infection with PRRSV.

weakening PRRSV-induced apoptosis. The antiviral activity of Matrine was reported on hepatitis B virus (Ye et al., 2007) and coxsackie B virus (Liu et al., 2003). These results suggested that Matrine may be a perspective and broad-spectrum antiviral candidate. The N protein is a multifunctional and the most abundant viral protein expressed in infected cells, constituting 20–40% of the protein content of a virion (Meulenberg, 2000). The most prom-

inent role of N protein is its interaction with the viral RNA in the assembly of the viral nucleocapsid. Cysteine residues and disulphide bridge formation acted on RNA binging (Dokland, 2010; Jourdan et al., 2012). Furthermore, N protein could be involved in inhibition of host protein synthesis owing to the concentration in the nucleolus which may affect ribosome assembly or function, thus leading to inhibition of protein synthesis (Suárez, 2000). Previous study speculated that N protein may affect transcriptional regulation in PRRSV infected cells because of its interacting with a HIC (human I-mfa domain-containing protein) homologue, a zinc-finger binging transcriptional regulator (Dokland, 2010). In our study, we demonstrated that Matrine inhibited N protein expression in a dose dependent manner. And the effect of inhibition was more obvious when cells were simultaneously infected with PRRSV and Matrine, suggesting Matrine may inactivate PRRSV directly and interfere with PRRSV assembly via at least inhibiting N protein expression. The exact mechanisms of such as action at a molecular interaction level are remained to be elucidated. There are a number of published studies indicated PRRSV could induce cell apoptosis (Miller and Fox, 2004). In our study, apoptotic cells and cleaved caspase-3 were examined during the first 24 h of infection and no difference in its expression relative to cell control was found, suggesting that major apoptosis-related genes including caspase-3 expression were not occurring within the first 24 h of infection (Miller and Fox, 2004). In our previous study, caspase-3 was not activated until 48 h of PRRSV infection (Wang et al., 2013). Further, Costers et al. used a specific substrate, DEVD-pNA to measure caspase-3 activity and found the level of caspase-3 activity was about 2.5-fold and 4.0-fold higher at 48 and 60 h post-infection, respectively, compared to uninfected control cells, suggesting PRRSV triggers caspase-3 activation after 48 h post-infection which confirmed our results (Costers et al., 2008). Moreover, in our studies, nearly 100% cells were infected with PRRSV at 48 h post-infection, but not all apoptotic signals of cells were detected by AnnexinV-PI double staining, suggesting apoptosis induction may not play a major role in PRRSV replication in vitro. Previous research indicated that activation of JNK signaling pathway is essential for PRRSV-mediated apoptosis but not a modulator of virus replication (Yin et al., 2012). In addition, GP5 of PRRSV and TNF-a may mediate PRRSV induced apoptosis (Choi and Chae, 2002; Suárez et al., 1996; Zhou and Zhang, 2012). In our study, Matrine inhibited PRRSV-induced cell apoptosis including at early and late stages of apoptosis and prevented caspase-3 activation. It is speculated that Matrine may interfere GP5 or/and TNF-a, resulting to inhibition of PRRSV induced apoptosis. Furthermore, Matrine could provide an alkaline environment which is hostile to PPRSV. Taking all these together, it is very likely that the observed anti-apoptotic effect of Matrine is attributed to its direct virucidal effect. In conclusion, Matrine possessed in vitro anti-PRRSV activity. Its antiviral effect might be attributed to four probable pathways: (1) directly inactive PRRSV; (2) attenuation of viral replication after its entry; (3) inhibit PRRSV induced apoptosis via preventing caspase3 activation; (4) interfere N protein expression. These results set a solid basis for further elucidating detailed antiviral mechanisms of Matrine. Acknowlegements This project was funded by the Key Scientific and Technological Grant from Shanxi Province (Grant No. 2010311047 and 20120311022-1). These experiments comply with the current laws of PR China. The authors have no conflict of interests.

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