Berberine inhibits hepatitis C virus entry by targeting the viral E2 glycoprotein

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Berberine inhibits hepatitis C virus entry by targeting the viral E2 glycoprotein Ting-Chun Hung , Alagie Jassey , Ching-Hsuan Liu , Chien-Ju Lin , Chun-Ching Lin , Shu Hui Wong , Jonathan Y. Wang , Ming-Hong Yen , Liang-Tzung Lin PII: DOI: Reference:

S0944-7113(18)30306-4 https://doi.org/10.1016/j.phymed.2018.09.025 PHYMED 52621

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Phytomedicine

Received date: Revised date: Accepted date:

21 April 2018 13 July 2018 3 September 2018

Please cite this article as: Ting-Chun Hung , Alagie Jassey , Ching-Hsuan Liu , Chien-Ju Lin , Chun-Ching Lin , Shu Hui Wong , Jonathan Y. Wang , Ming-Hong Yen , Liang-Tzung Lin , Berberine inhibits hepatitis C virus entry by targeting the viral E2 glycoprotein, Phytomedicine (2018), doi: https://doi.org/10.1016/j.phymed.2018.09.025

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PHYMED-D-18-00570.R1

Berberine inhibits hepatitis C virus entry by targeting the viral E2 glycoprotein

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Hui Wong , Jonathan Y. Wang , Ming-Hong Yen

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International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan International Master Program in Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

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Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

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Ting-Chun Hung , Alagie Jassey , Ching-Hsuan Liu , Chien-Ju Lin , Chun-Ching Lin , Shu

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CORRESPONDENCE (1) Liang-Tzung Lin, Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 11031, Taiwan, Tel: +886-2-2736-1661 ext. 3911, E-mail: [email protected]. (2) Ming-Hong Yen, Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, No. 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, Tel: +886-7-3121101 ext. 2665, E-mail: [email protected].

Running Title: Berberine inhibits HCV Entry Word Count: (Abstract) 212 words; (Introduction to Discussion) 4067 words Figures & Tables: 7 Figures

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ABSTRACT Background: Despite the advent of direct-acting antivirals (DAAs), HCV remains an important public health problem globally. There is at present no effective vaccine against the virus, and the DAAs in current use cannot prevent de novo infection, including in liver transplant setting

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wherein donor livers inevitably become re-infected. Developing inhibitors to HCV entry using nature-derived small molecules may help to expand/complement the current treatment options. Purpose: In this study, we explored the effect of the plant alkaloid berberine (BBR) on HCV early viral entry.

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Methods: Cell culture-derived HCV (HCVcc), viral pseudoparticles bearing HCV glycoproteins (HCVpp), and entry-related assays were employed to assess BBR’s bioactivity. Molecular docking was used to predict BBR-HCV glycoproteins interaction, and the compound’s antiviral activity was confirmed against HCVcc infection of primary human hepatocytes (PHHs).

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Results: BBR specifically impeded HCVcc attachment and entry/fusion steps without inactivating the free virus particles or affecting the expression of host cell entry factors and post-

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entry viral replication. BBR also effectively inhibited infection by viral pseudoparticles expressing HCV E1/E2 glycoproteins and molecular docking analysis pointed at potential interaction with

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HCV E2. Finally, BBR could suppress HCVcc infection of PHHs. Conclusions: We identified BBR as a potent HCV entry inhibitor, which merits further

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evaluation particularly for use in transplant setting against graft re-infection by HCV.

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Keywords: HCV; berberine; alkaloid; natural product; antiviral; entry inhibitor

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ABBREVIATIONS BBR, berberine; CC50, 50% cytotoxic concentration; CD81, cluster of differentiation 81; CHKV, Chikungunya virus; CLDN1, claudin-1; DAA, direct-acting antiviral; EC50, 50% effective concentration; EGFR, epidermal growth factor receptor; GAGs, glycosaminoglycans; HCC,

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hepatocellular carcinoma; HCV, hepatitis C virus; HCVcc, cell culture-derived HCV; HCVpp, HCV pseudoparticles; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IFN-α, interferon-alpha; LDLR, low density lipoprotein receptor; NPC1L1, Niemann-Pick C1-Like 1; OCLN, occludin; PHH, primary human hepatocyte; PUG, punicalagin; RLU, relative light units;

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SEM, standard error of the means; SI, selective index; SR-BI, scavenger receptor class B type I.

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INTRODUCTION Hepatitis C Virus (HCV) is an important pathogen infecting approximately 170-300 million people globally and predisposing the majority to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). Consequently, HCV represents an important

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public health issue and is a leading cause of liver transplantation in most industrialized countries (de Oliveria Andrade et al., 2009).

The 9.6 kb genome of the flavivirus encodes a single polyprotein that is processed by host and viral proteases to produce 3 structural (Core, E1, and E2) and 7 nonstructural (p7, NS2,

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NS3, NS4A, NS4B, NS5A, and NS5B) proteins upon entry via clathrin pit-mediated endocytosis into human hepatocytes. The pre-requisite for successful HCV entry involves the wellorchestrated interactions of the viral particle and its E1 and E2 glycoproteins with a series of host cell factors at the cell membrane, including glycosaminoglycans (GAGs), cluster of

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differentiation 81 (CD81), low density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1), occludin (OCLN), epidermal growth factor receptor (EGFR),

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and Niemann-Pick C1-Like 1 (NPC1L1) (Douam et al., 2015). There is currently no effective vaccine to protect against HCV infection. Recent development of direct-acting antivirals (DAAs)

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that specifically target the viral non-structural proteins have much improved the previous decade’s PEGylated interferon-alpha (IFN-α)-based regimens, achieving sustained virological

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response rates to almost 90% in the most difficult-to-treat genotypes (Feeney and Chung, 2014). Nonetheless, several challenges remain, including the high cost, development of resistant

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mutants, and usage in the difficult-to-treat populations such as HCC patients and patients with other co-morbidities (Zoulim et al., 2015). Identifying novel lead compounds to complement or expand the current therapeutic options could help improve management of hepatitis C populations and address these challenges. Entry inhibitors are an additional class of antivirals which could be useful in restricting viral infection spread in the absence of an effective vaccine. In hepatitis C, such inhibitor class 4

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could be particularly useful in transplant setting to prevent de novo HCV infection of donor liver allograft, which are inevitably re-infected almost immediately in HCV-positive patients (Verna and Brown, 2008). Although neutralizing antibodies against surface receptor/co-receptors have been shown to impede HCV entry (Ball et al., 2014), small molecules derived from natural

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products targeting HCV entry have recently been extensively explored (Burnouf et al., 2017). However, there are no FDA-approved entry inhibitors yet, highlighting the need to continuously develop novel leads.

Plant secondary metabolites such as terpenes, flavonoids, and alkaloids represent an

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excellent source of antiviral discovery. The alkaloids are pharmacologically active, nitrogencontaining basic compounds and include the compound berberine (BBR), which has been previously explored for antiviral effects against other viruses such as Chikungunya virus (CHKV), Semliki Forest virus, Sindbis virus (Varghese et al., 2016), enterovirus 71 (Wang et al., 2017),

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herpes simplex virus (HSV) (Chin et al., 2010), and human immunodeficiency virus (HIV) (Bodiwala et al., 2011). However, the effects of BBR on HCV entry remain unknown.

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In order to expand the scope of novel antivirals particularly those targeting HCV entry, we examined in this study the influence of BBR treatment against HCV infection. Using various

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entry-related assays, our results indicated that BBR could efficiently inhibit HCV infection, particularly during the early viral entry stages. In addition, using viral pseudoparticles bearing

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HCV E1/E2 glycoproteins, we demonstrated that BBR could impede the HCVpp infection, and further analysis using molecular docking revealed potential interaction of BBR with the HCV E2.

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Finally, treatment with BBR suppressed HCV particle infection of primary human hepatocytes, suggesting that the small molecule merit to be further explored as lead or antiviral for prophylaxis/treatment of hepatitis C.

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MATERIALS AND METHODS Cell culture, reagents, and virus production The human hepatoma Huh-7.5 cells were grown in Dulbecco’s modified Eagle's medium (DMEM; GIBCO-Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 1%

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gentamycin, and 1% amphotericin B in a 5% CO 2 incubator at 37°C. The cell culture-derived HCV (HCVcc; kindly provided by Dr. Charles M. Rice, Rockefeller University, USA) expressing a secreted Gaussia luciferase reporter upon productive infection was produced as previously described (Liu et al., 2017), through electroporation of Huh-7.5 cells with run-off RNA transcripts

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generated from the genotype 2a Jc1FLAG2 (p7-nsGluc2A) construct (Lin et al., 2013). BBR (≥98% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in ddH2O. Punicalagin was kindly provided by Dr. Ta-Chen Lin (Central Taiwan University of Sciences and Technology, Taiwan) and IFN-α was obtained from Sigma. For all virus infection experiments, a

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basal medium containing 2% FBS with antibiotics was used.

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Cytotoxicity analysis

Huh-7.5 cells were seeded in 96-well plates at a density of 1 x 104 cells/well overnight and

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treated with various concentrations of BBR (0, 2, 5, 10, 20, 50, and 100 μM) for 72 h at 37°C. At the end of the incubation, cells were washed twice with PBS and analyzed by the XTT

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2011).

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cytotoxicity assay to determine percent (%) cell viability as previously described (Lin et al.,

Antiviral assay For the anti-HCV activity of BBR, Huh-7.5 cells were seeded in 96-well plates at a confluence of 1 x 104 cells/well overnight after which various concentrations of the compound (0, 2, 5, 10, 20, 50, and 100 μM) were added simultaneously to the cells at the time of viral infection and

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incubated for 72 h at 37°C. The anti-HCV activity was determined using the BioLux™ Gaussia Luciferase Assay Kit (New England Biolabs; Pickering, ON, Canada) with a luminometer (Promega; Madison, WI, USA), and the HCV infectivity was expressed as log10 relative light

Synchronized infection assay on early viral entry

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units (log10 RLU).

The synchronized infection assay on early viral entry was carried out as described previously (Lin et al., 2013). Specifically, BBR was tested for impact on cell-free virus particles, viral

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attachment, and viral entry/fusion. Luciferase activity for all conditions was determined after the end of the 72 h-incubation as described earlier.

Viral replication assay

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Huh-7.5 cells were electroporated with 10 μg Jc1FLAG2 (p7-nsGluc2A) RNA before seeding in 12-well plates for a 24 h-recovery incubation as previously described (Hsu et al., 2015). The

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cells were subsequently washed with PBS and overlaid with complete medium containing BBR for 48 h. Luciferase activity in the supernatant reflecting viral replication/translation was then

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determined as described earlier.

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Pretreatment analysis

The pre-treatment analysis was performed as previously described (Lin et al., 2011), by

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pretreating Huh-7.5 cells with BBR for 24 h before PBS wash and infection with HCVcc (MOI 0.01). Luciferase activity was determined at the end of the 72 h-incubation period as described earlier.

B18R IFN-antagonist assay

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The B18R IFN-antagonist assay followed a previously described method (Lin et al., 2015). Huh7.5 cells seeded at 1 x 10 4 cells/well were treated with or without B18R (10 ng/ml; eBioscience; San Diego, CA, USA) for 2 h. Cells were then infected with HCVcc (MOI = 0.01) in the presence or absence of BBR or the positive control IFN-α (800 IU/ml). At 3 days post-infection, luciferase

presence or absence of IFN antagonism from B18R.

Western blotting analysis of HCV entry factors

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reporter activity was determined to compare the specific treatment’s antiviral effect in the

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Huh-7.5 cells (5 × 105 cells/well in 6-well plates) were treated with BBR for at least 24 h followed by extracting cellular proteins for analysis using standard Western blotting technique. Immunoblotting was carried out using the specific antibodies: anti-CD81 (1:1000; BD Biosciences, San Jose, CA, USA), anti-SR-BI (1:1000; Abcam, Cambridge, UK), anti-Claudin-1

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(1:1000; Invitrogen), anti-OCLN (1:200; Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1:20000; Santa Cruz Biotechnology, Dallas, Texas, USA) primary antibodies, and

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anti-mouse (1:5000; Invitrogen) or anti-rabbit (1:2500; Sigma) secondary antibodies. Images

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were obtained using an UVP chemiluminescence imaging system (UVP; Upland, CA, USA).

Viral pseudoparticle infectivity analysis

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Production of HCV pseudotypes based on loading HCV E1/E2 glycoproteins onto a lentiviral core containing reporter gene has been previously reported (Bartosch and Cosset, 2009), and a

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similar strategy was employed. The plasmid pJ6CE1E2 was constructed by synthesizing the HCV genotype 2a (HC-J6CH strain; NC_009823) complete core followed by E1/E2 glycoproteins and cloning into pcDNA3.1 expression vector (Life Technologies; Grand Island, NY, USA) using standard cloning techniques. The inclusion of the complete core has been previously observed to enhance production of genotype 2 HCV pseudoparticles (Shukla et al., 2010). To generate HCV pseudoparticles (HCVpp) bearing HCV E1/E2, Env-defective HIV 8

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proviral construct that encodes firefly luciferase in place of the Nef gene (pNL.Luc.EnvR; generously provided by Dr. Éric A. Cohen, Institut de Recherches Cliniques de Montréal, Canada) (Connor et al., 1995) was co-transfected with pJ6CE1E2 in 293T cells using OMNIfect™ in vitro transfection reagent (transOMIC Technologies Inc.; Huntsville, AL, USA)

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(Garcia et al., 2009). Virus-containing supernatant was harvested, clarified, passed through a 0.45 μm filter, and concentrated by 25% (v/v) polyethylene glycol prior to use. For HCVpp infectivity analysis, Huh-7.5 cell monolayers in 12-well plates were challenged with the HCVpp in the presence or absence of test compound for 2 h at 37°C. Following infection, the cells were

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washed twice with PBS and then incubated in basal medium for 3 days. At the end of the incubation, the cells were harvested and analyzed for luciferase reporter activity, which was generated upon successful entry of the HCVpp, using the Firefly Luciferase Assay kit (Promega) and a luminometer. Data was plotted as the percentage (%) of relative light units (RLU) from

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Molecular docking analysis

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test compound treatment relative to control.

The HCV (genotype: 1a strain H77) E1 N-terminus and E2 x-ray diffraction structures were

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gathered from the Protein Data Bank (PDB: 4UOI and 4MWF, respectively) (El Omari et al., 2014; Kong et al., 2013). BBR’s 3-D conformation was obtained from Pubchem (PMCID: 2353).

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Molecular docking was performed using Autodock Vina (Scripps, LLC; La Jolla, CA, USA) to predict the free energy associations of bound conformations between ligand and protein targets

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(Trott and Olson, 2010). Bound conformations were examined using the PyMol Molecular Graphics System (Version 1.7.4, Schrödinger, LLC; Portland, OR, USA).

Antiviral analysis on HCVcc infection of primary human hepatocytes Infection of primary human hepatocytes (PHHs) using HCVcc was carried out as previously described (Hsu et al., 2015). Briefly, commercially obtained PHHs (GIBCO-Invitrogen) were 9

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cultured on collagen I-coated plates using primary hepatocytes supplements and William’s medium E (GIBCO-Invitrogen) according to the manufacturer’s protocol. After 48 h of incubation, PHHs were challenged with HCVcc infection (MOI 0.1) in the presence of test compound or control for 3 h at 37°C, then washed and incubated in the primary hepatocytes culturing medium.

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Subsequently, the supernatant was collected following 72 h of incubation and analyzed for luciferase activity.

Statistical analysis

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All data are expressed as means ± standard error of the means (SEM). Statistical analysis was performed using multiple t-tests. A P value of less than 0.05 (P < 0.05) was considered

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statistically significant. All experiments were conducted with at least three independent repeats.

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RESULTS BBR inhibits HCV infection at non-cytotoxic concentrations BBR (Fig. 1A) has been previously suggested to possess antiviral activities against a variety of viruses, but whether or not the alkaloid possess similar antiviral activities against HCV remains

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unknown. To explore the anti-HCV activity of BBR, we infected Huh-7.5 cells with HCVcc reporter virus in the presence of various concentrations of the drug (0, 1, 5, 10, 20, 50, and 100 μM) for 72 h. A cytotoxicity analysis was concomitantly carried out on the cells at the same compound treatment concentrations. Results indicated that BBR exhibited a dose-dependent

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antiviral activity against HCVcc infection for up to a concentration of 20 μM without exhibiting significant cytotoxic effect (Fig. 1B). The 50% cytotoxic concentration (CC50), 50% effective concentration (EC50), as well as the selective index (SI), were determined to be 82.75 ± 0.27 μM, 7.87 ± 1.10 μM, and 10.51, respectively. Given that BBR at 20 μM significantly inhibited HCV

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infection without inducing significant cytotoxicity, we therefore used this concentration for all our

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subsequent experiments.

BBR targets the early steps of HCV viral entry

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We next sought to investigate the mechanism(s) involved in BBR’s antiviral effect against HCV infection, including targeting viral entry. To test for this hypothesis, we performed a

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synchronized infection assay on the early viral entry steps of HCV (Fig. 2A) to determine whether the drug could inactivate free viral particles, inhibit viral attachment, and/or block viral

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entry/fusion. Punicalagin (PUG), a tannin known to exert inhibitory effect against HCV particles in all three steps of the early viral entry (Lin et al., 2013), was included as positive control. To delineate the effect of BBR on cell-free virus particles, BBR was pre-incubated with HCVcc inoculum for 3 h before dilution to ineffective concentration of the drug; this dilution prevents meaningful influence from the drug on the subsequent steps of the experiment. The resulting inoculum was then used to challenge the cells before further incubation for luciferase reporter 11

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analysis. Our results showed no significant difference in the luciferase activity in the presence or absence of BBR, suggesting that the alkaloid does not inactivate/neutralize the cell-free viral particles (Fig. 2B). To examine the impact of BBR on viral attachment, pre-chilled Huh-7.5 cells were

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simultaneously inoculated with BBR and the HCVcc inoculum at 4°C (Fig. 2A), which allows for viral particle binding to the host cells while precluding viral fusion/entry. The cells were subsequently washed with PBS and incubated for another 3 days before luciferase analysis. Results in Fig. 2B demonstrated a significant decrease in HCVcc luciferase activity in the

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presence of BBR, indicating that BBR robustly blocked HCVcc attachment to host cells.

To evaluate the drug influence on viral fusion/entry, Huh-7.5 cells were pre-bound with HCVcc particles at 4°C before treatment with medium containing BBR and shifting the temperature to 37°C to facilitate viral entry (Fig. 2A). The resulting luciferase reporter activity

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after 3 days of incubation demonstrated a significant reduction in the presence of BBR, denoting that BBR could as well inhibit HCV entry/fusion, albeit to a moderate degree compared to its

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effect in the viral attachment scenario (Fig. 2B).

Together, these results showed that while BBR had little influence on the free virus

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particles, the drug could effectively target and inhibit HCV infection at the attachment and

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entry/fusion steps.

BBR’s antiviral effect does not implicate inhibition of HCV replication post entry or

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induction of antiviral response To examine whether BBR has additional impact on the viral life cycle post entry, we electroporated Huh-7.5 cells with Gaussia luciferase-tagged full-length HCV genome to bypass the entry step, and then incubated these cells with medium containing BBR for 72 h before assessing the accumulated reporter signal generated upon viral replication. As shown in Fig. 3A, no significant difference was observed in the post-entry viral replicative life cycle with or without 12

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BBR treatment. In contrast, IFN-α treatment, which served as a positive control, significantly reduced the HCV infection (Fig. 3A). To rule out other influence(s) of BBR, for instance in modulating the host cell against the HCV infection, we investigated the impact of BBR pre-treatment on the hepatoma cells for 24 h

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prior to HCVcc inoculation. Results showed no significant difference of HCV infectivity in cells with or without BBR pre-treatment, and both demonstrated similar luciferase signal output (Fig. 3B). To further examine the possibility of BBR in inducing the host cell antiviral response, we tested BBR’s treatment in the presence of the IFN antagonist B18R, a known inhibitor of type I

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IFN response (Alcami et al., 2000). Again, BBR’s antiviral activity against the HCV infection was unaltered with or without the presence of the IFN antagonist B18R, which, on the other hand, could rescue the viral infection from the IFN-α treatment (Fig. 3C). These observations therefore suggested that BBR’s antiviral effect did not involve modulating the host cell including in

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inducing IFN immune response.

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Expression of key host cell entry factors to HCV infection are unaltered by BBR’s treatment

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Since BBR specifically targeted HCV entry, we further explored the underlying mechanism of its antiviral bioactivity. Blocking viral entry can be mediated by influencing the expression of viral

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entry receptors/co-receptors or interfering with the functions of HCV E1/E2 glycoproteins. To clarify how BBR inhibits the early steps of HCV infection, we next conducted a Western blot

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analysis on hepatoma cells treated with or without BBR for 24 h and examined for alteration in protein expression of key HCV entry factors including SR-BI, CD81, CLDN1, and OCLN (Douam et al., 2015). Results showed no difference in the protein expression of these HCV receptors/coreceptors whether or not BBR was used (Fig. 4). Further incubation to 72 h with the compound yielded a similar observation (data not shown). These results suggest that BBR treatment does not modulate the HCV host cell entry factors. 13

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BBR inhibits E1/E2 glycoproteins-mediated HCVpp infection Based on the above observation, we looked into the other possibility of BBR potentially targeting and interfering with the HCV envelope glycoproteins. For this purpose, we generated lentivirus-

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based pseudoparticles expressing a luciferase reporter gene and the HCV envelope glycoproteins E1 and E2 (HCVpp) (Bartosch and Cosset, 2009). Monolayers of Huh-7.5 cells were challenged with the HCVpp in the presence or absence of BBR and luciferase reporter signals generated upon successful viral entry was determined after 3 days of incubation. PUG

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treatment was included as positive control. As shown in Fig. 5, BBR treatment significantly reduced the HCVpp infection, suggesting the HCV envelope glycoproteins E1 and E2 as potential targets of BBR’s antiviral action.

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BBR interacts with HCV envelope glycoprotein E2

To substantiate on the above findings, we performed molecular docking analysis on HCV E1

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and E2 glycoprotein crystal structures (El Omari et al., 2014; Kong et al., 2013) to predict whether BBR interacts with these viral glycoproteins. Although the average binding energy was

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similar between BBR and each of the glycoproteins (E1: 6.6 kcal/mol versus E2: 6.4 kcal/mol for all potential docking frames), no polar contacts were observed between the compound and the

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E1 N-terminus (data not shown). Our analysis returned a single polar contact from BBR to the Ser599 residue on the E2 monomer with the highest binding energy of 6.7 kcal/mol (Fig. 6). This

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location is on average 19, 20, and 17 Å away from the conserved Cys569, Cys581, and Cys585 residues, respectively, which are part of where the disulfide bridges form between E1 and E2 in the heterocomplex (Freedman et al., 2016). More importantly, the region of BBR-E2 interaction is in close proximity (about 11 Å) to the hypervariable region 3 (HVR3; residues 431-466), an area documented to participate in CD81 receptor binding and viral entry (Freedman et al., 2016; Troesch et al., 2006). Additionally, all other frames from the analysis docked BBR in close 14

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proximity to the HVR3 region on E2 (data not shown). Taken together, the above analysis points to the HCV E2 as a potential target of BBR’s antiviral activity against the early HCV entry steps.

BBR blocks HCV infection of primary human hepatocytes

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Finally, to assess BBR’s value for further development as a candidate antiviral or lead compound, we tested its inhibitory effect against HCVcc infection of primary human hepatocytes (PHHs), which are a gold standard for hepatic in vitro culture models. PHHs were concurrently challenged with HCVcc and BBR for 72 h followed by luciferase reporter analysis. In agreement

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with our preceding findings, BBR also efficiently inhibited HCVcc infection of the PHHs (Fig. 7). Altogether, our results suggest that BBR robustly impedes HCV entry steps including attachment and fusion/entry by potentially interacting with the HCV E2 glycoprotein, and further

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supports its development as a starting point candidate entry inhibitor against HCV.

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DISCUSSION Plant secondary metabolites represent an important source of antiviral drug discovery against viral infections. BBR, an alkaloid reported to possess antiviral activities against many viruses, is shown for the first time in this report to also harbor antiviral function in restricting HCV infection

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of both Huh-7.5 cells and PHHs by potentially interacting with the HCV E2 glycoprotein. Our discovery of BBR’s anti-HCV activity adds to the growing list of natural entry inhibitory molecules against the virus such as the green tea polyphenol epigallocatechin-3-gallate (ECGC) (Ciesek et al., 2011), the butenolide (4R,6S)-2-Dihydromenisdaurilide (Chung et al., 2016b),

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loliolide derived from Phyllanthus urinaria (Chung et al., 2016a), the terpenoid saikosaponin b2 (Lin et al., 2015), and the flavonoid ladanein (Haid et al., 2012), amongst others. Collectively, our data point to a prominent antiviral role of BBR against HCV entry and identify the small molecule as a new class of natural product with encouraging anti-HCV bioactivity.

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Owing to its applicability in both prophylaxis and therapeutic treatment, entry inhibitors are an important class of antiviral agents, particularly when the virus infection is without an

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effective vaccine. In the case of hepatitis C, HCV entry inhibitors could potentially be used to prevent re-infection of liver grafts in transplant setting (Burnouf et al., 2017). Previous

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experience with the HIV highly active antiretroviral therapy (HAART) has demonstrated the advantage to include entry inhibitors (e.g. enfuvirtide) with other antiretroviral class of agents to

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intensify antiretroviral treatment and combat multidrug resistance (Haqqani and Tilton, 2013). Given that the current hepatitis C DAAs mostly target the HCV nonstructural proteins to inhibit

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viral replication, combining DAAs with entry inhibitors could allow a multi-pronged approach to target HCV and mitigate the development of drug resistance, which is an important cause of DAA failures (Feeney and Chung, 2014). Intriguingly, the use of HCV entry inhibitors in combination with DAAs has been shown to induce synergistic effects against HCV infection (Xiao et al., 2014). Thus, our discovery that BBR suppresses HCV entry could serve as an

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impetus for further development and exploration of the small molecule as an entry inhibitor that could be used in combination with existing DAAs. Our HCVpp assay (Fig. 5) and molecular docking analysis (Fig. 6) led to the hypothesis that BBR potentially targets the HCV E2 glycoprotein in mediating its antiviral effect. The HCV

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E2 is primarily responsible for preventing immature viral fusion as well as stabilizing the viral attachment (Freedman et al., 2016). Binding of certain residues in the E2 HVR3 region neutralizes CD81 receptor interaction and viral entry (Freedman et al., 2016; Troesch et al., 2006). For these reasons, we postulate that the targeting of BBR to HCV E2, which was

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predicted to be at Ser599 near the HVR3 region (Fig. 6), may explain its antiviral effect in antagonizing HCV entry steps. Interestingly, although BBR potentially targets to E2, our results from Fig. 2B showed that BBR was not able to inactivate cell-free HCV particles despite efficiently inhibiting particle attachment and the subsequent entry/fusion. A possible explanation

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is that the only single polar contact of BBR to Ser599 on E2 may not be strong enough to allow the natural compound to stay bound and inactivate the viral particles. However, due to the

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proximity to HVR3, which participates in receptor engagement for mediating HCV entry (Freedman et al., 2016; Troesch et al., 2006), the targeting of E2 by BBR may be sufficient to

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block virus attachment and the consequent entry steps to the host cell. This deduction could explain BBR’s most pronounced antiviral effects against the virus binding step and not in

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inactivating/neutralizing free virus particles (Fig. 2B). Of note, while our prediction shows proximity to the E2 HVR3 region, we could not rule out other potential binding sites of BBR on

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the E2 glycoprotein, since the available E2 structure 4MWF is missing several residues including regions 482-494, 385-411 (HVR1), and 475-481 (HVR2) (The UniProt, 2017). Further analysis using a full E1/E2 complex crystallography structure would allow a more accurate analysis of BBR with the HCV glycoproteins. Finally, our analysis also showed a potential influence of BBR on HCV viral fusion/entry, albeit less prominent compared to its inhibitory effect on virus binding (Fig. 2B). This is 17

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indicative that BBR may also possess antiviral activity against the post-attachment viral entry steps, such as the fusion of the viral membrane with the endosomal membrane (Burnouf et al., 2017). How this occurs via BBR’s interaction with E2 or through additional mechanism of action(s) remains to be clarified. It is also interesting to note that while our analysis

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demonstrated an impact of BBR specifically on HCV entry, the compound has been observed to exert inhibitory effect throughout the early and late stages of CHKV (Varghese et al., 2016) and against the viral replicative phase of HSV (Chin et al., 2010) infections. This suggests that BBR may possess various modes of action as a broad-spectrum antiviral agent. Future in-depth

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studies would be necessary to help define the complete antiviral profile of BBR and its possible application under different viral infection scenarios, including hepatitis C.

CONCLUSIONS

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In summary, we demonstrated in this study that BBR robustly inhibits HCV entry, specifically blocking HCV attachment and entry/fusion steps, possibly by transiently interacting with the

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HCV E2 glycoprotein. Our results support BBR as lead compound or candidate drug for the

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development of entry inhibitors in the prophylaxis/treatment of hepatitis C.

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ACKNOWLEDGEMENTS The authors would like to thank Drs. Charles M. Rice, Éric A. Cohen, and Ta-Chen Lin for reagents, and Shun-Pang Chang, Chueh-Yao Chung, and Chia-Lin Li for experimental support.

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FUNDINGS This study was supported in part by funding from the Ministry of Science and Technology of Taiwan (MOST106-2320-B-038-021 to L.T.L. and MOST105-2320-B-037-008 to C.C.L.).

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

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CONFLICTS OF INTEREST

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T.C.H. and L.T.L. conceived and designed the experiments. T.C.H., A.J., C.H.L., C.J.L., S.H.W., and J.Y.W. performed the experiments. M.-H.Y. and L.T.L supervised all research. T.C.H., A.J.,

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C.H.L., C.J.L., C.C.L., S.H.W., J.Y.W., M.H.Y., and L.T.L. analyzed the data. T.C.H., A.J., C.H.L., M.H.Y., and L.T.L. wrote and edited the paper. All authors contributed to

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reagents/materials/technical support to this study.

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FIGURE LEGENDS Fig. 1. Cytotoxicity and antiviral activity of BBR against HCV infection. (A) Chemical structure and molecular weight of BBR. (B) Huh-7.5 cell cytotoxicity and anti-HCV activity of BBR. IFN-α (800 IU/ml) served as positive control. All data represent means ± SEM from 3

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independent experiments performed in triplicate.

Fig. 2. Effect of BBR on early viral entry of HCV infection. (A) A schematic of synchronized

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infection analysis of BBR’s effect on HCV early viral entry steps. Cells were washed with PBS between each incubation period. (B) Influence of BBR (20 μM) treatment on free virus particles,

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viral attachment, and viral entry/fusion. Punicalagin (PUG; 50 μM) served as positive control.

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Data shown are means ± SEM from 3 independent experiments; *** = P < 0.001 compared to

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“Virus Only” treatment.

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Fig. 3. Effect of BBR on HCV replication and induction of antiviral response. (A) Viral replication assay in the presence BBR (20 μM) treatment. A schematic showing the

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experimental conditions of the assay is indicated. (B) Pre-treatment analysis of BBR (20 μM) on HCV infectivity. The associated schematic depicts the experimental procedure. (C) B18R IFNantagonist assay on BBR treatment of HCV infection. B18R (10 ng/ml); BBR (20 μM). IFN-α (800 IU/ml) served as positive control. Data shown are means ± SEM from 3 independent experiments; *** = P < 0.001, ns = not significant.

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Fig. 4. Influence of BBR on the expression levels of HCV host cell receptors. Cells were pre-treated with BBR (20 μM) for 24 h before harvesting total protein and probing for the indicated proteins. Representative blots from 3 independent experiments are shown. β-actin

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served as loading control.

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Fig. 5. Impact of BBR on hepatoma infection by retroviral pseudoparticles bearing HCV E1/E2 glycoproteins. Luciferase reporter-tagged HCVpp was used to challenge Huh-7.5 cells

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in the presence or absence of BBR treatment (20 μM). Punicalagin (PUG; 50 μM) served as

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positive control. All data represent means ± SEM from 3 independent experiments; *** = P <

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Fig. 6. Molecular docking analysis of BBR’s interaction with HCV E2. The HCV E2 monomer (white) in relation to the viral membrane is shown with BBR (yellow), the HVR3 region (red), and the conserved cysteine residues (teal); the demarcated area in blue is shown as the

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zoomed panel on the right. Polar contact from BBR to E2 is indicated by black dash in the

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zoomed panel.

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Fig. 7. Effect of BBR on HCV infection in PHHs. PHHs were inoculated with HCVcc in the presence or absence of BBR treatment (20 μM). Data shown are means ± SEM from 3

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independent experiments; *** = P < 0.001 compared to “Virus Only” treatment.

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Graphical Abstract

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