Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells

Antiviral Research 174 (2020) 104704

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Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells

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Weibo Liaoa,b,1, Xin Liua,c,1, Quanlue Yanga,c, Huifang Liuc, Bingyu Lianga,c, Junjun Jianga,c, Jiegang Huanga, Chuanyi Ningc, Ning Zangc, Bo Zhouc, Yanyan Liaoa,c, Jingzhao Chena,b, Li Tiana,b, Wenzhe Hod, Abu S. Abdullahe, Lingbao Kongf, Hao Lianga,c,∗∗∗, Hui Chena,b,∗∗, Li Yea,c,∗ a

Guangxi Key Laboratory of AIDS Prevention and Treatment & Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China c Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China d Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA, 19140, USA e Boston University School of Medicine, Boston Medical Center, Boston, MA, 02118, USA f Institute of Pathogenic Microorganism, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, 330045, Jiangxi, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Deguelin Hepatitis C virus Autophagy Beclin1

Aims: Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. Methods: HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. Results: Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and timedependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3BII levels, the conversion of LC3B–I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. Conclusions: Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.

1. Introduction Hepatitis C virus (HCV) infection remains one of the major public health problems in the world. Up to 2017, there are approximately 71 million individuals worldwide infected with HCV, representing about 1% of the world's population (Spengler, 2018). Each year, more than 400 000 deaths which have increased by 22% since 2000 are related to HCV infection. And the late complications developing from chronic

hepatitis C such as cirrhosis and hepatocellular carcinoma (HCC) are the major causes (Calvaruso et al., 2018). In the past few years, a few novel antivirals against HCV have been developed. Several direct-acting antivirals (DAAs) which target different nonstructural proteins of HCV have been approved by FDA successively since 2011, including HCV NS3/4A protease inhibitors telaprevir and simeprevir, NS5A inhibitors daclatasvir and ledipasvir, and NS5B polymerase inhibitors sofosbuvir and dasabuvir (Spengler, 2018).

∗

Corresponding author. Guangxi Medical University, Shuangyong Road 22, Nanning, Guangxi, 530021, China. Corresponding author. Guangxi Medical University, Shuangyong Road 22, Nanning, Guangxi, 530021, China. ∗∗∗ Corresponding author. Guangxi Medical University, Shuangyong Road 22, Nanning, Guangxi, 530021, China. E-mail addresses: [email protected] (H. Liang), [email protected] (H. Chen), [email protected] (L. Ye). 1 These authors contributed equally to this paper. ∗∗

https://doi.org/10.1016/j.antiviral.2020.104704 Received 24 August 2019; Received in revised form 15 December 2019; Accepted 2 January 2020 Available online 07 January 2020 0166-3542/ © 2020 Elsevier B.V. All rights reserved.

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In addition, updated triple antiviral regimen [pegylated-interferon (PEG-IFN) with ribavirin (RBV) mostly plus sofosbuvir] has increased the sustained virologic response rate (SVR) and shortened treatment time in HCV patients (Bunchorntavakul et al., 2018; Jakobsen et al., 2017). Nevertheless, drug resistance (Esposito et al., 2016; Ikram et al., 2017), extreme bradycardia (Renet et al., 2015), and the other concerns including narrow genotype specificity (Moradpour et al., 2016), as well as the high cost (Rosenthal and Graham, 2016) implied the need to develop new anti-HCV drugs with different anti-HCV mechanisms, to overcome the shortcomings and provide a new component for combined therapy for radical cure of HCV infection. Natural compounds, which have a diversity of structures, provide a large reservoir for screening anti-HCV agents with new structure and novel mechanisms. Deguelin, a natural product isolated from Mundulea sericea (Leguminosae) and certain other plants (Ganesh Yerra et al., 2013), has been demonstrated anti-proliferation activities and/or induce apoptosis in many human cancers, such as lung cancer (Xu et al., 2015b), colon cancer (Kang et al., 2012), hepatocellular cancer (Lee et al., 2008) and breast cancer (Mehta et al., 2013). In addition, some publications suggest that deguelin has the wound healing, anti-inflammatory (Lodhi et al., 2016), and antimicrobial activities (Lee et al., 2005). However, the antiviral activity of deguelin has not been reported so far. A recent study shows that deguelin exhibits an activity to inhibit cellular autophagy in pancreatic cancer cells (Xu et al., 2017), which sparks our interest in investigation of relationship among deguelin, HCV infection, and cellular autophagy. Autophagy has been widely recognized as a promoting factor that closely related to HCV replication (Dreux et al., 2009; Wang et al., 2014, 2015). On the one hand, autophagy machinery is required for effective HCV replication. Autophagosomes may act as a scaffold for intracellular membrane-associated replication of HCV and initiation of HCV genomic replication (Fahmy and Labonte, 2017; Tanida et al., 2009). Furthermore, several autophagic proteins including Beclin-1, Atg5 have the activity to promote HCV productive infection (Guevin et al., 2010; Shrivastava et al., 2012). On the other hand, HCV can subvert the autophagic pathway, for example, inducing autophagy vesicles in hepatocytes, in favor of its replication (Hansen et al., 2017; Huang et al., 2014; Medvedev et al., 2017). Therefore, autophagy has been considered a potential target for the development of novel anti-HCV drugs (Fabri et al., 2011; Panigrahi et al., 2015; Subauste, 2009). In this study, we investigated whether deguelin has the activity of inhibiting HCV replication in human hepatoma cells through its impact on cellular autophagy as well as mechanism(s) involved.

Fig. 1. Cytotoxic effect of deguelin on Huh7 cells. Huh7 cells were treated with deguelin at indicated concentrations for 72 h. The cell viability was assessed by ATP assay. The shown data are the mean ± SD of three independent experiments. The p value was calculated by Student's t-test (*, p < 0.05; **, p < 0.01).

incubator with 5% CO2. Generation of HCV JFH-1 infectious virus and in vitro infection followed the previously described (Wakita et al., 2005). Infection of Huh7 cells with HCV JFH-1 was carried out at a multiplicity of infection (MOI) of 0.1. 2.3. Deguelin treatment Huh7 cells in 24-well or 12-well plates were incubated in the presence of deguelin at different concentrations for 24 h–120 h. Tunicamycin-treated Huh7 cells were used as a positive control for inducing autophagy in cells. 2.4. Beclin1 overexpression and silencing expression Beclin1 overexpression was carried out by transfection of plasmid containing Beclin1, pCMV-myc-Beclin1 (p-BEclin1) (Qiagen, Shanghai, China). Silencing Beclin1 expression was performed by transfection of siRNA to Huh7 cells, which contains specific sequence (TAAGTAATG GAGCTGTGAGTT) against Beclin1 (Qiagen, Shanghai, China). Plasmid and siRNA transfections were performed with Lipofectamine3000 according to manufacturer's instructions. 2.5. RNA extraction and real-time RT-PCR

2. Materials and methods Total cellular RNA was extracted from cells with TaKaRa MiniBEST Universal RNA Extraction Kit (Takara, Dalian, China). The cDNA was synthesized using Quantitect kit (Qiagen, Shanghai, China) and then subjected to real-time RT-PCR with Real-Time SYBR Green PCR Master Mix (Takara, Dalian, China). Primer sequences for target genes are shown in Supplementary Table 1.

2.1. Reagents Deguelin, tunicamycin (Tu), choroquine (CQ) and secondary antibodies (horseradish peroxidase conjugated goat-anti-rabbit IgG, goatanti-mouse IgG) for Western blot were purchased from Sigma-Aldrich China (Shanghai, China). Anti-β-actin was purchased from Abcam Trading China (Shanghai, China). Anti-Beclin1, anti-HCV core antibody and Lipofectamine3000 were purchased from Thermo Fisher Scientific China (Shanghai, China). Anti-LC3, anti-p62 and anti-ATG16L1were purchased from Cell Signaling Technology China (Shanghai, China).

2.6. Western blot Cells were washed twice with cold PBS and then lysed in radio immunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor cocktail (PMSF) and PhosSTOP (Roche, USA). The protein concentrations were detected by DC protein kit (Beyotime Institute of Biotechnology, Shanghai,china). Proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were incubated with various primary antibodies, followed by secondary horseradish peroxidase-conjugated antibodies. The immunoreactive bands were visualized by SuperSignal West Pico chemiluminescence substrate (Thermofisher, USA). Densitometric analysis of blots was performed by Image J software (National Institutes of Health,

2.2. Huh7 cells, HCV JFH-1 and virus infection The human hepatoma Huh7 cell line and HCV JFH-1 virus were kindly provided by Dr. Wenzhe Ho (Temple University, Philadelphia, USA). Huh7 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 nM nonessential amino acids (NEAA), 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were maintained in a 37 °C 2

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Fig. 2. Inhibition of HCV replication by deguelin treatment in HCV JFH-1-infected Huh7 cells. Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. (A, B) The dose effect of deguelin on HCV replication. The infected cells at day 3 postinfection were treated with deguelin at indicated concentrations for 72 h. (C, D) The time course effect of deguelin on HCV replication. The infected cells at day 3 postinfection were treated with deguelin at 0.4 μM for different times. (A, C) The levels of intracellular HCV RNA in deguelin-treated or untreated control cells, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without deguelin treatment, which was defined as 1, respectively). (B, D). Representative Western blot images show HCV core protein expression in deguelin-treated or untreated cells. (B, D) The densitometric intensities of HCV core and β -actin bands were quantified by image J software. The relative HCV core/β-actin ratios were calculated and shown as the fold of control (without deguelin treatment, which was defined as 1, respectively). The data shown in Fig. 2 are the mean ± SD of the results of three independent experiments. The p value was calculated by Student's ttest (*, p < 0.05; **, p < 0.01).

Bethesda, MD, USA). The values were normalized to those of control βactin. The primary antibodies used for Western blot were as follows: anti-β-actin (1:2000), anti-Beclin1 (1:1000), anti-HCV core (1:2000), anti-LC3 (1:1000), anti-p62 (1:1000), anti-ATG16L1 (1:1000).

2.8. Statistical analysis Experimental data were subjected to Student's t -test of difference between control and treatment groups. Data were expressed as mean ± standard deviation (SD) of at least triplicate experiments. Statistical analyses were performed with SPSS 20.0, and statistical significance was defined as p < 0.05.

2.7. Autophagosome formation by fluorescence microscope Transient green fluorescent protein (GFP)-LC3-expressing Huh7 cells were generated using lentivirus-mediated GFP-LC3 overexpression (Fang et al., 2017; Li et al., 2017). A lentiviral vector containing GFPLC3 fusion gene and lentivirus were provided by Genechem Company (Shanghai, China). Autophagy was assessed using GFP-LC3 redistribution in cells that was detected by an inverted fluorescence microscope (Nikon Ti-s). The number of GFP-LC3-positive dots per cell was determined in 3 independent experiments (30 cells were counted per experiment).

3. Results 3.1. Cytotoxic effect of deguelin on Huh7 cells We firstly investigated the cytotoxic effect of deguelin on cell viability in Huh7 cells (human hepatoma cell line). The ATP test (CellTiterGlo® Luminescent Cell Viability Assay) shows that there was no significant difference in cell viability between deguelin-treated cells with concentrations at 0.4 μM or lower and control group (Fig. 1). However, the growth of Huh7 cells was significantly inhibited when the concentration of deguelin reached 0.8 μM or higher (Fig. 1). In the 3

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Fig. 3. The effect of deguelin treatment on expression levels of two markers of autophagy, LC3 and p62, in Huh7 cells, tunicamycin (Tu)-treated or HCV-infected cells in the absence of choroquine (CQ). (A, B) Huh7 cells were treated or untreated with deguelin (0.4 μM) for 72 h; (C, D) Huh7 cells treated with tunicamycin (5 μg/mL) were simultaneously treated or untreated with deguelin (0.4 μM) for 72 h; (E, F) Huh7 cells were infected with HCV JFH-1 at an MOI of 0.1. At day 3 postinfection, the HCV-infected cells were treated or untreated with deguelin (0.4 μM) for 72 h. After deguelin treatment, the cellular RNA and proteins were extracted for real time RT PCR and Western blot analysis. (A, C, E) The levels of LC3 mRNA in deguelin-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without deguelin treatment under different conditions, which was defined as 1, respectively). (B, D, F) Representative Western blot images show LC3B–I, LC3B-II and p62 protein levels in deguelin-treated or untreated cells under different conditions. (B, D, F) Quantitative assessment of LC3B–I, LC3B-II, LC3B-II/LC3B–I, and p62 at protein levels in deguelin-treated or untreated cells under different conditions. The densitometric intensities of LC3B–I, LC3B-II, p62, and β -actin bands were quantified by image J software. The relative LC3B–I/β -actin, LC3B-II/β–actin, LC3B-II/LC3B–I, and p62/β-actin ratios were calculated and shown as the fold of control (without deguelin treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 3 are the mean ± SD of the results of three independent experiments. The p value was calculated by Student's t-test (*p < 0.05, **p < 0.01).

form (LC3-II), which is associated with autophagosomal membranes. In addition, p62, a marker of autophagic flux, is a cargo receptor for autophagic degradation of ubiquitinated targets that binds directly to LC3-II, and is exclusively degraded during autophagy. The decreased levels of p62 could be observed when autophagy is activated. Because the decrease in LC3B-II level may reflect impaired induction of autophagy or enhanced autophagic degradation, to distinguish these two possibilities, cells were treated with a late stage autophagy inhibitor choroquine (CQ), which could block the fusion of autophagosomes with lysosomes and prevent lysosomal degradation. In the absence of choroquine (CQ) (Fig. 3), at LC3 mRNA level, no significant effect (p > 0.05) was observed in deguelin-treated Huh7 cells compared with untreated control cells (Fig. 3A). Accordingly, no significant difference (p > 0.05) was observed in LC3B–I protein level between two groups (Fig. 3B). However, a significant impact of deguelin on LC3B-II expression in Huh7 cells was observed. The level of LC3B-II (LC3B-II/β-actin) as well as conversion of LC3B–I to LC3B-II (LC3B-II/LC3B–I) in deguelin-treated cells were significantly decreased when compared with those in untreated control cells (Fig. 3B). Meanwhile, expression of p62 significantly increased in deguelin-treated cells (Fig. 3B). Similarly, under the condition of tunicamycin treatment (an autophagy inducer) (Fig. 3C and D) or HCV JFH-1 infection (Fig. 3E and F), decreased expression of LC3B-II (LC3B-II/β-actin, LC3B-II/LC3B–I) and increased expression of p62 were also observed in deguelin-treated cells, compared with deguelin-untreated tunicamycin-treated cells

following experiments, the treatment concentrations of deguelin were selected at 0.4 μM or lower, to avoid the occurrence of cytotoxicity which could further influence HCV replication in cells. 3.2. Deguelin inhibits HCV JFH-1 replication in Huh7 cells We then investigated whether deguelin has an anti-HCV activity in HCV JFH-1-infected Huh7 cells. Deguelin treatment of cells significantly inhibited intracellular HCV RNA expression (Fig. 2A and C). When the cells were treated with deguelin at 0.4 μM for 72 h–120 h, the level of intracellular HCV RNA decreased to less than 25% of that in control cells. The inhibitory effect deguelin on HCV replication was confirmed at the protein level (Fig. 2B and D). The levels of HCV core protein in deguelin-treated cells were significantly lower than those in control cells (Fig. 2B and D). Either at HCV RNA level or at core protein level, the inhibitory effect of deguelin on HCV replication was observed in a dose-dependent and time-dependent manner (Fig. 2). 3.3. Deguelin treatment suppresses cellular autophagy in Huh7 cells To examine whether deguelin affects cellular autophagy in Huh7 cells, we firstly investigated whether deguelin modulates the expression of two markers of autophagy, microtubule-associated protein 1 light chain 3 (LC3) and p62. LC3 has two forms, LC3-I and LC3-II. During autophagy, the soluble form (LC3-I) of LC3 is converted to a lipidated 4

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Fig. 4. In the presence of choroquine (CQ), the effect of deguelin treatment on LC3 and p62 levels in Huh7 cells, tunicamycin (Tu)-treated or HCV-infected cells. After 2 h pre-incubation with the autophagy inhibitor CQ (25 μM), (A, B) Huh7 cells were treated or untreated with deguelin (0.4 μM) for 72 h; (C, D) Huh7 cells treated with tunicamycin (5 μg/mL) were simultaneously treated or untreated with deguelin (0.4 μM) for 72 h; (E, F) HCV-infected cells at day 3 postinfection were treated or untreated with deguelin (0.4 μM) for 72 h. After deguelin treatment, the cellular RNA and proteins were extracted for real time RT PCR and Western blot analysis. (A, C, E) The levels of intracellular LC3 mRNA in deguelin-treated or untreated cells under different conditions, with normalization to corresponding GAPDH mRNA level, are expressed as the fold of control (without deguelin treatment under different conditions, which was defined as 1, respectively). (B, D, F) Representative Western blot images show LC3B–I, LC3B-II, p62 protein levels in deguelin-treated or untreated cells under different conditions. (B, D, F) Quantitative assessment of LC3B–I, LC3B-II, LC3B-II/LC3B–I, and p62 at protein level in deguelin-treated or untreated cells under different conditions. The densitometric intensities of LC3B–I, LC3B-II, p62, and β -actin bands were quantified by image J software. The relative LC3B–I/β-actin, LC3B-II/β-actin, LC3B-II/LC3B–I and p62/βactin ratios were calculated and shown as the fold of control (without deguelin treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 4 are the mean ± SD of the results of three independent experiments. The p value was calculated by Student's t-test (*p < 0.05, **p < 0.01).

3.4. Deguelin treatment inhibits autophagosome formation

(Fig. 3D) or deguelin-untreated HCV-infected cells (Fig. 3F), respectively. Therefore, in the absence of CQ, these data imply that deguelin treatment suppresses cellular autophagy, even under the condition of HCV infection. CQ treatment could lead to greatly increased expression of LC3B-II and p62 compared with those in CQ-untreated cells (Fig. 3B vs. Fig. 4B; Fig. 3D vs. Fig.4D; Fig. 3F vs. Fig. 4F), because CQ inhibits the degradation function of lysosomes, and thus results in the accumulation of LC3B-II and p62. In the presence of CQ, deguelin had little effect on expression levels of LC3 mRNA, LC3B–I protein, and p62 protein (p > 0.05), but led to decreased expression of LC3B-II (LC3B-II/βactin, LC3B-II/LC3B–I), compared with deguelin-untreated cells (Fig. 4A and B). As autophagy inducers, tunicamycin treatment (Fig. 4D) or HCV infection (Fig. 4F) displayed its inducing function more significantly by greatly increased expression of LC3B-II in the presence of CQ. Nevertheless, under the condition of tunicamycin treatment (Fig. 4D) or HCV infection (Fig. 4F), deguelin also exhibited its inhibitory effect on expression of LC3B-II (LC3B-II/β-actin, LC3B-II/ LC3B–I), indicating that deguelin does have the activity to inhibit cellular autophagy, more importantly, it inhibits the upstream event(s) at early stage of autophagy, due to CQ (inhibitor at late stage of autophagy) did not interfere deguelin's inhibitory effect on LC3B-II expression (Fig. 4).

To confirm the finding based on measurement of LC3B-II and p62 levels, the impact of duguelin on autophagic flux was also examined in autophagosome formation using GFP-LC3 fluorescent assay. Fig. 5 shows the numbers of GFP-LC3-positive dots that representing the amount of autophagosomes in different groups. In control cells (without deguelin treatment, tunicamycin treatment, and HCV infection) or deguelin-treated cells, only few GFP-LC3-positive dots were observed (Fig. 5A, B, 5G), however, in deguelin-treated cells, the numbers of dots significantly decreased (Fig. 5G). In tunicamycin-treated or HCV-infected cells (2 h pre-treatment with 25 μM CQ), much more GFP-LC3positive dots were observed (Fig. 5C, E, 5G), indicating both tunicamycin and HCV infection could induce formation of autophagosomes. When tunicamycin-treated or HCV-infected cells were treated by deguelin, a significant decrease in numbers of GFP-LC3-positive dots were observed (Fig. 5D, F, 5G), indicating that deguelin treatment could inhibit the autophagosome formation induced by tunicamycin or HCV infection. 3.5. The effect of deguelin treatment on the expression of autophagy-related genes Since we have seen that the effect of deguelin on autophagy involves in early stage of autophagy, we further investigated whether 5

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Fig. 5. The effect of deguelin treatment on autophagosome formation in Huh7 cells, tunicamycin (Tu)-treated or HCV-infected cells. After 2 h pre-incubation with the autophagy inhibitor CQ (25 μM), deguelin-treated and -untreated Huh7 cells were observed under the fluorescent microscopy. (A, B) Huh7 cells were treated or untreated with deguelin (0.4 μM) for 72 h. (C, D) The tunicamycin (Tu)-treated (5 μg/mL) Huh7 cells were treated or untreated with deguelin (0.4 μM) for 72 h; (E, F) HCV JFH-1-infected Huh7 cells at day 3 postinfection were treated or untreated with deguelin (0.4 μM) for 72 h. Magnification 200 × . (G) GFP-LC3–positive dots per cell were determined under the fluorescent microscopy (30 cells were counted per experiment). Data shown in Fig. 5G is mean ± SD of the results of three independent experiments. The p value was calculated by student's t-test (*, p < 0.05).

3.7. Silencing expression of Beclin1 enhances the inhibitory effect of deguelin on HCV replication

deguelin affects mTOR pathway and BECN1-PIK3C3 pathway, the most important negative and positive regulators for autophagy in mammalian cells, respectively (Zhang et al., 2015). The expression of key factors of these two pathways, including mTOR, Beclin1, ATG4, ATG12, ATG16L1, AKT, ULK1 and GABARAPL1 were measured upon deguelin treatment using real time RT-PCR in HCV-uninfected Huh7 cells (Fig. 6A) or HCV JFH-1-infected cells (Fig. 6C). The results show that the mRNA expression of Beclin1 and ATG16L1 was significantly downregulated by deguelin treatment in HCV-uninfected or -infected Huh7 cells (Fig. 6A and C). Western blot assay confirmed the decreased expression of Beclin-1 at protein level (Fig. 6B and D). However, ATG16L1 protein in deguelin-treated cells had no significant difference either in HCV-uninfected or in HCV-infected Huh7 cells when compared with those in deguelin-untreated cells (Fig. 6B and D). Therefore, Beclin-1 might involve in the inhibitory effect of deguelin on cellular autophagy.

We also investigated the role of Beclin1 in deguelin's inhibition of HCV replication through silencing Beclin1 expression via transfection of specific siRNA against Beclin1 (in the absence of CQ). Western blot analysis confirmed the silencing efficiency of si-Beclin1 on Beclin1 expression in Huh7 cells (Fig. 8A). In deguelin-untreated or -treated cells, silencing expression of Beclin1 decreased HCV core protein expression, compared with the control cells, respectively (Fig. 8B). The autophagic flux examination shows that silencing expression of Beclin1 decreased the expression of LC3B-II (LC3B-II/β-actin) and conversion of LC3B–I to LC3B-II (LC3B-II/LC3B–I), but enhanced p62 expression (Fig. 8B). In deguelin-treated cells, silencing expression of Beclin1 also led to decreased expression of LC3B-II and increased expression of p62, along with enhanced inhibitory effect of deguelin on HCV replication (Fig. 8B). Thus, combined data of overexpression (Fig. 7) and silencing expression (Fig. 8) indicate that Beclin1 does play a role in deguelin's inhibitory effect on cellular autophagy as well as HCV replication.

3.6. Overexpression of Beclin1 weakens the inhibitory effect of deguelin on HCV replication Next, we investigated whether over-expressed Beclin1 alters deguelin's inhibitory effect on autophagy and HCV replication (in the absence of CQ). Transfection of pCMV-myc-Beclin1 (p-Beclin1) to Huh7 cells effectively increased Beclin1 protein expression (Fig. 7A). In deguelin-untreated or -treated cells, overexpression of Beclin1 could increase or partially restore HCV replication, respectively, evidenced by the increased level of HCV core protein in pBeclin1-transfected cells compared with blank plasmid-transfected cells (Fig. 7B). The examination of autophagic flux shows that overexpression of Beclin1 enhanced the expression of LC3B-II (LC3B-II/β-actin) and conversion of LC3B–I to LC3B-II (LC3B-II/LC3B–I), but decreased p62 expression (Fig. 7B). In deguelin-treated cells, overexpression of Beclin1 also led to increased expression of LC3B-II and decreased expression of p62, along with partially restoring HCV replication inhibited by deguelin treatment (Fig. 7B).

4. Discussion In the present study, for the first time, we found that a natural compound, deguelin, has an anti-HCV activity in human hepatoma Huh7 cells. Deguelin is one of major natural rotenoids obtained from Mundulea sericea (Leguminosae) and certain other plants such as Derris Trifoliate (a Chinese herb), Cubé resin, or root extracts from plant species such as Lonchocarpus utilis and Lonchocarpus urucu (Boyd and Han, 2016). In the previous studies, it has been well known for application of deguelin as insecticide, acaricide, piscicide, and fishing poison due to its inhibition of cellular respiration via inhibition of NADH/ ubiquinone oxidoreductase (complex I) of the mitochondrial electron transport chain (ETC) (Vrana et al., 2013). Recently, deguelin has also been investigated for its potential use as chemopreventive and chemotherapeutic medicine due to its anticancer activity in a variety of 6

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Fig. 6. The effect of deguelin on expression of autophagy-related genes in Huh7 cells or HCV JFH-1-infected Huh7 cells. Huh7 cells (A, B) or HCV-infected Huh7 cells at day 3 post infection (C, D) were cultured in the presence or absence of deguelin (0.4 μM) for 72 h. The cellular RNA and proteins were extracted for real-time RT-PCR and Western blot analysis. (A, C) The mRNA levels of mTOR, Beclin1, ATG4A, ATG12, ATG16L1, Akt, ULK1, and GABARAPL1 in deguelin-treated or -untreated cells under condition of HCV noninfection or infection, with normalization to corresponding GAPDH mRNA, are expressed as the fold of control (without deguelin treatment under different conditions, which was defined as 1, respectively). (B, D) Representative Western blot images show Beclin1 and ATG16L1 protein expression in deguelin-treated or untreated cells under different conditions. (B, D) Quantitative assessment of Beclin1 and ATG16L1 protein expression. The densitometric intensities of Beclin1, ATG16L1, and β-actin bands were quantified by Image J software. The relative Beclin1/β-actin and ATG16L1/β-actin ratios were calculated and shown as the fold of control (without deguelin treatment under different conditions, which was defined as 1, respectively). The data shown in Fig. 6 are the mean ± SD of the results of three independent experiments. The p value was calculated by student's t-test (*p < 0.05, **p < 0.01).

(Figs. 3–5), indicating that deguelin does have the activity to directly suppress cellular autophagy and then inhibit HCV replication. In this study, tunicamycin, a known autophagy inducer, is used as positive control for observation of inducing autophagy. It can induce ER stress and then active autophagy through accumulating unfolded proteins in cell endoplasmic reticulum (ER), with the similar mechanism as HCV infection induces (Ma et al., 2016; Yorimitsu and Klionsky, 2007). Our mechanism research indicated that down-regulated expression of Beclin1 by deguelin accounts for its inhibitory effect on autophagy and HCV replication (Figs. 6–8), which is not the same as shown in the earlier study on pancreatic cancer cells (Xu et al., 2017). This report shows the suppression of autophagy by deguelin occurs in late stage of autophagy, and deguelin inhibits autophagosome maturation. This discrepancy can be explained by that different cells may possess different response mechanism upon external stimuli. More importantly, the concentrations of deguelin they used were rather high (5μM–50μM), within this range deguelin not only suppresses autophagy but also induces apoptosis simultaneously (Xu et al., 2017). Whereas in our study, the concentration of deguelin is very low (≤0.4 μM), and we did not observe the occurrence of cytotoxicity including apoptosis (data not shown). Beclin1, a component of the class III phosphatidylinositol 3-kinase (PI3K) complex, is required for the initiation of the autophagosome formation in autophagy. As the homolog of the yeast Atg6 gene, Beclin1 is the first identified operational gene which participates in autophagy

cancer types. Mechanism investigation manifested that deguelin treatment results in cell cycle arrest (Wang et al., 2013), induction of apoptosis (Baba et al., 2015), inhibition of the activities of NF-κB and PI3K/Akt signaling pathways (Kang et al., 2012). However, in our study, we found that deguelin treatment has little effect on cellular apoptosis or PI3K/Akt pathway (data not shown). One reason for this discrepancy is that the concentration of deguelin used in our study is very low, to avoid the occurrence of cytotoxicity, which, of course, is not likely to affect cell cycle or induce apoptosis. This study shows that the inhibitory effect of deguelin on HCV replication involves in its suppression on cellular autophagy. Autophagy is a universal cellular mechanism response to the stress of nutrient limitation, and its primary functions include maintaining homeostasis, preserving the balance among the synthesis, degradation, and recycle the cellular components (Mizushima and Komatsu, 2011). It also plays a diverse role in host defense against invading pathogens (Mizushima, 2005). For HCV, a number of studies have highlighted the importance of cellular autophagy to HCV replication (Taguwa et al., 2011; Tanida et al., 2009). Therefore, it is easy to understand that the suppression of autophagy by deguelin leads to inhibition of HCV replication. Deguelin treatment also inhibits autophagy in HCV-infected cells, one possibility is that deguelin inhibits HCV replication firstly and then results in suppression of autophagy, due to HCV infection is a kind of autophagy inducer. Nevertheless, our study found that deguelin could suppress autophagy in HCV-uninfected and tunicamycin-treated Huh7 cells 7

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Fig. 7. Over-expression of Beclin1 weakens the inhibitory effect of deguelin on HCV replication. HCV JFH-1-infected Huh7 cells at day 3 postinfection were transfected with plasmids pCMV-myc-Beclin1 (pBeclin1) or blank plasmid, and the transfection maintained for 48 h. (A) Overexpression efficiency of pBeclin1 was determined by Western blot. The densitometric intensities of Beclin1and β-actin bands were quantified by Image J software. The relative Beclin1/β-actin ratios were calculated and shown as the fold of control (without plasmid transfection, which was defined as 1). (B) Over-expression of Beclin1 on effect of deguelin on autophagy and HCV replication. Plasmid-transfected cells were treated with or without deguelin (0.4 μM), and the treatment was maintained for 48 h. (B) A representative Western blot image shows HCV core, LC3B–I, LC3B-II, and p62 protein levels. (B) Quantitative assessment of HCV core, LC3B-II, LC3B-II/LC3B–I, and p62 at protein level. The densitometric intensities of HCV core, LC3B–I, LC3B-II, p62 and β-actin bands were quantified by image J software. The relative HCV core/β-actin, LC3B-II/β-actin, LC3B-II/LC3B–I, and p62/β-actin ratios were calculated and shown as the fold of control (without deguelin treatment/blank plasmid transfection, which was defined as 1). The data shown in Fig. 7 are the mean ± SD of the results of three independent experiments. The p value was calculated by Student's t-test (*, p < 0.05, **, p < 0.01).

potentially be further developed as an important autophagy inhibitor to overcome shortcoming of current HCV therapy.

(Kondo and Kondo, 2006). It contains four functional structure domains: Bcl-2–homology-3 (BH3), central coiled coil domain (CCD), evolutionarily conserved domain (ECD) and nuclear output structure domain (Cao and Klionsky, 2007). These domains can form “Beclin1 Autophagy Complex” by combining with multiple proteins to execute related functions. In addition to the role in initiation of autophagy, Beclin1 can also indirectly enhance HCV replication through suppression of type I IFN signaling pathway. Previous reports have indicated that Beclin1 suppresses RIG-I-MAVS axis, by which negatively regulates type I IFN signaling. Beclin1 deficiency promotes the activation of the IFN-β (Jin et al., 2016; Xu et al., 2015a). Thus, down-regulated expression of Beclin1 by deguelin may further contribute to inhibition of HCV replication through enhancement of type I IFN response in hepatic cells. In summary, we provide the evidence for the first time, showing that deguelin, a natural product derived from Mundulea sericea (Leguminosae) and certain other plants could efficiently inhibit HCV replication in vitro. The anti-HCV activity of deguelin was found to be dependent on its down-regulation of Beclin1 expression and thus suppression cellular autophagy, which has been considered as an effective novel strategy against HCV infection. Therefore, deguelin could

Declaration of competing interest The authors have no conflicts of interest to declare. Acknowledgements This work was supported by National Natural Science Foundation of China (NO. 81460305, NO.31560050, and NO.81860655); Guangxi Scientific and Technological Development Project, China (NO. Gui Ke Gong 14124003–1); Guangxi University “100-Talent” Program & Guangxi university innovation team and outstanding scholars program, China (NO. Gui Jiao Ren 2014[7]). Innovation Project of Guangxi Graduate Education, China (NO.YCSW2018118); Guangxi Bagui Scholar, China (to Junjun Jiang). We would also like to thank Dr. Yiping Li at Zhongshan School of Medicine, Sun Yat-sen University, China and Dr. Gang Long at Institute Pasteur of Shanghai, Chinese Academy of Sciences, China, for their guidance and technical support for this work. 8

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Fig. 8. Silencing expression of Beclin1 enhances the inhibitory effect of deguelin on HCV replication. HCV JFH-1-infected Huh7 cells at day 3 postinfection were transfected with si-Beclin1 or scramble siRNAs, and the transfection maintained for 48 h. (A) Silencing efficiency of si-Beclin1 was determined by Western blot. The densitometric intensities of Beclin1and β-actin bands were quantified by Image J software. The relative Beclin1/β-actin ratios were calculated and shown as the fold of control (without siRNA transfection, which was defined as 1). (B) Silencing expression of Beclin1 on effect of deguelin on autophagy and HCV replication. siRNA-transfected cells were treated with or without deguelin (0.4 μM), and the treatment was maintained for 48 h(B) A representative Western blot image shows HCV core, LC3B–I, LC3B-II, and p62 protein levels. (B) Quantitative assessment of HCV core, LC3B-II, LC3B-II/LC3B–I, and p62 at protein level. The densitometric intensities of HCV core, LC3B–I, LC3B-II, p62 and β-actin bands were quantified by image J software. The relative HCV core/β-actin, LC3B-II/β-actin, LC3B-II/LC3B–I, and p62/β-actin ratios were calculated and shown as the fold of control (without deguelin treatment/scramble siRNA transfection, which was defined as 1). The data shown in Fig. 8 are the mean ± SD of the results of three independent experiments. The p value was calculated by Student's t-test (*, p < 0.05, **, p < 0.01).

Abbreviations HCV JFH-1 LC3 HCC GFP CQ DAAs PEG-IFN SVR RT-PCR RIPA DMEM FBS NEAA MOI ER ATG PI3K

ETC BH3 CCD ECD RIG-I

hepatitis C virus Hepatitis C Virus Japanese fulminant hepatitis −1 Microtubule-associated proteins-light chain3 hepatocellular carcinoma green fluorescent protein chloroquine directly acting antivirals pegylated-interferon sustained virologic response rate real-time immunoprecipitation assay buffer Dulbecco's modified Eagle's medium fetal bovine serum non-essential amino acids multiplicity of infection endoplasmic reticulum autophagy-related gene phosphatidylinositol 3-kinase

electron transport chain Bcl-2–homology-3 central coiled coil domain evolutionarily conserved domain retinoic acid-inducible gene I

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