Anti-influenza A Virus Activity of Dendrobine and Its Mechanism of

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Anti-influenza A Virus Activity of Dendrobine and Its Mechanism of Action Richan Li,† Teng Liu,† Miaomiao Liu,† Feimin Chen,† Shuwen Liu,*,† and Jie Yang*,† †

Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China S Supporting Information *

ABSTRACT: Dendrobine, a major component of Dendrobium nobile, increasingly draws attention for its wide applications in health care. Here we explore potential effects of dendrobine against influenza A virus and elucidate the underlying mechanism. Our results indicated that dendrobine possessed antiviral activity against influenza A viruses, including A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 H274Y (H1N1), and A/Aichi/2/68 (H3N2) with IC50 values of 3.39 ± 0.32, 2.16 ± 0.91, 5.32 ± 1.68 μg/ mL, respectively. Mechanism studies revealed that dendrobine inhibited early steps in the viral replication cycle. Notably, dendrobine could bind to the highly conserved region of viral nucleoprotein (NP), subsequently restraining nuclear export of viral NP and its oligomerization. In conclusion, dendrobine shows potential to be developed as a promising agent to treat influenza virus infection. More importantly, the results provide invaluable information for the full application of the Traditional Chinese Medicine named “Shi Hu”. KEYWORDS: Dendrobium nobile, dendrobine, antiviral, viral nucleoprotein, influenza A virus

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INTRODUCTION Influenza, an infectious disease primarily caused by influenza A virus (IAV), continues to raise worldwide concerns due to its high morbidity and significant mortality. With the antigenic drift/shift, adaptation, and genetic reassortment, highly virulent strains may appear unexpectedly, resulting in epidemics locally or pandemics worldwide and seriously threatening public health, as well as the economy.1,2 For example, the pandemic of 1918−1919 H1N1 influenza killed an estimated 50 million persons.3 In addition, the influenza H1N1 (2009) virus rapidly spread to 214 countries around the world, causing the deaths of at least 18,000 people.4 Recent reports have demonstrated that avian influenza H5N1 and H7N9 viruses may cross the species barrier and cause infection in humans,5,6 suggesting that we would face more serious situations in preventing outbreaks of influenza viruses in the future. Therefore, the prophylaxis and early treatment for these devastating pandemic viruses have become important, urgent global problems. Currently, two main strategies against influenza are available, which are vaccination and anti-influenza drugs. Vaccination is an optimal strategy to reduce the burden of disease in the prevention of influenza infection. However, to match antigenic changes of virus, effective vaccination may require constant reproduction, implying that it seems almost impossible to produce efficient and timely vaccines to control influenza outbreaks.7,8 For this reason, anti-influenza drugs present the first line of protection against the virus during a pandemic, particularly in the early stages.9,10 Two classes of anti-influenza drugs that target M2 channel or neuraminidase (NA) are approved for clinical prevention and treatment of influenza until now. However, resistant variants have continued to emerge from patients after treatment regardless of both classes of drugs, especially amantadine and rimantadine.11−13 Besides, prolonged use of the above anti-influenza agents exhibited side © 2017 American Chemical Society

effects. For example, adamantane is known to have potential central nervous system (CNS) side effects.13 Thus, the ongoing threat of resistance and adverse effects of these drugs highlight the pressing demand for developing new anti-influenza agents with novel mechanisms. Since ancient times, herbal plants have distinctive superiority compared to modern medicine and play an essential role in the treatment of illness.14 Numerous herbs and their bioactive ingredients, such as alkaloids, saponins, flavonoids, polyphenols, tannins, and glucosides, receive growing attention as an important source of therapeutic agents.15 To date, several natural products and botanical extracts against influenza infection have been reported. For instance, phlorotannins of the brown alga Ecklonia cava showed inhibition activity against influenza virus.16 Polyphyllasaponin I isolated from Parispolyphyla possessed an inhibitory effect on IAV both in vitro and in vivo.17 Gallic acid extracted from Rubus coreanus seed showed anti-influenza activity via disrupting viral particles.18 We previously demonstrated quercetin effectively inhibited IAV infection via interaction with viral hemagglutinin (HA) protein.19 Here, we found dendrobine, a main active component isolated from Dendrobium nobile, could significantly inhibit IAV replication in vitro. The plant D. nobile, also named “Shi Hu” in Chinese, is a perennial herb belonging to the orchid family and widely distributed in the tropical and subtropical regions of Europe, Asia, and Oceania. In the Chinese Pharmacopoeia (National Pharmacopoeia Committee, 2015), fresh or dried stem of D. nobile is recorded as one of the original materials of “Shi Received: Revised: Accepted: Published: 3665

January 19, 2017 April 14, 2017 April 18, 2017 April 18, 2017 DOI: 10.1021/acs.jafc.7b00276 J. Agric. Food Chem. 2017, 65, 3665−3674

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Journal of Agricultural and Food Chemistry

Antiviral Assay and Microscopy. To determine the antiviral activity of dendrobine, confluent MDCK cells were infected with the virus at a multiplicity of infection (MOI) of 0.01 at 37 °C for 1 h. Subsequently, dendrobine of non-cytotoxic concentrations was added to the cells after washing away the unabsorbed virus with PBS, and the cells were cultured for another 48 h. At the end of the culture, the MTT-based assay as previously described was assessed for the antiviral activity of dendrobine. The cytopathic effect (CPE) in virus-infected cells was observed through microscopy. Plaque Assay. Confluent monolayers of MDCK cells were inoculated with A/Aichi/2/68 (H3N2) strain at an MOI of 0.01 for 1 h at 37 °C. After removing the unbound virus, the cells were cultivated in 1.5 mL of serum-free MEM (2×) containing 1 μg/mL TPCK-trypsin (Sigma-Aldrich), 2% agar (Sigma-Aldrich), and different concentrations of dendrobine for 72 h as previously described.28 The effect of dendrobine on viral plaque formation was determined by the number of plaques. Western Blotting. MDCK cells were infected with A/Aichi/2/68 (H3N2) strain (MOI = 0.01) for 1 h, followed by the addition of dendrobine at serial concentrations. After 24 h of incubation, the cells were lysed and standardized for protein content. Then the sample was resolved by 10% SDS-PAGE. HA and β-actin were detected using primary anti-HA monoclonal antibody (1:200 dilution; Sino Biological, Beijing, China) or anti-β-actin monoclonal antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA) as control overnight at 4 °C, followed by the anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 dilution, Cell Signaling Technology). Finally, chemiluminescent signals were developed with Lumiglo reagent (Cell Signaling Technology) and exposed to X-ray film (Fujifilm Europe GmbH, Dusseldorf, Germany). Quantitative Real-Time PCR. The levels of viral genes were analyzed using quantitative real-time PCR as described previously.29 Briefly, the infected MDCK or A549 cells were treated with dendrobine at serial concentrations for 24 h. Then total RNA of cells was extracted by TRIzol reagent and reverse transcribed into cDNA using the primers listed in Table S1. Quantitative real-time PCR was performed by two-step PCR amplification standard procedure in an ABI7500 system (Applied Biosystems, Foster, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The relative expressions of HA gene were measured by a classical 2−ΔΔCT method using 7500 software. Moreover, the gene expression was also measured at 24, 48, and 72 h post-infection (pi) for observing the duration of treatment of dendrobine after multiple rounds of replication. Ribavirin was used as a positive control. Additionally, intracellular NP viral RNA (vRNA) in MDCK cells within 6 h pi was determined by quantitative real-time PCR to assess the inhibitory effect of dendrobine on viral genome replication.30 Briefly, the virus fluid was discarded and cells were cultured in medium with or without dendrobine after virus absorption. Zanamivir was used as a negative control. At 3 and 6 h pi, cells were lysed, and the extracted vRNA was reverse transcribed into cDNA using the IAVspecific RT primer (uni-12; 5′-AGCAAAAGCAGG-3′). Then cDNA was used for quantitative real-time PCR as described above. All reactions were performed in triplicate. Immunofluorescence Microscopy. Immunofluorescence microscopy was performed as described previously with simple alterations.31 Briefly, the MDCK or A549 cells grown on coverslips or in 48-well plates with 80% confluency were infected with the virus at an MOI of 1.0 or 0.01. The cells were washed and treated with dendrobine or zanamivir at 37 °C for 24 h. The cells were fixed for 10 min with 4% paraformaldehyde and then blocked for 1 h with 3% albumin from bovine serum (BSA) at room temperature. Antiinfluenza A virus nucleoprotein (NP) antibody (1:250 dilution, Santa Cruz, Dallas, TX, USA) was added to the cells, followed by fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:250 dilution, Santa Cruz). After counterstaining with 4,6-diamidino-2-phenylindole (DAPI), the samples were subjected to an confocal laser scanning microscope (Olympus FluoView FV1000, Tokyo, Japan) for 6 h pi and

Hu”, which has been used for medicinal purposes for more than 2000 years in China and has become an increasingly popular medical and health product now.20 In the daily diet, fresh or dried D. nobile can be eaten without further processing. The fresh stem is always used in soup or steeped wine after being washed and chopped. The soup and wine with Dendrobium can be helpful to strengthen body immunity.21 Moreover, the washed fresh Dendrobium can be chewed directly; it tastes sweet and is slightly sticky. Compared to the fresh stem, the dried stem is generally added into herbal tea, known as “liángchá” in Chinese, which is very popular in China.22 Nowadays, D. nobile has also been added to a Chinese health care product catalog and European Union novel foods catalog. D. nobile possesses multiple pharmacological activities due to its many chemical components, mainly including alkaloids, sesquiterpenoids, aromatic compounds, and polysaccharides.23,24 Among these active components, many studies have demonstrated alkaloids have unexpected effects in the antiviral field.15 Dendrobine is the major active alkaloid from the stem of Dendrobium. The quality control and discrimination of D. nobile are determined by the content of dendrobine, because it is higher than other types of “Shi Hu”.25 Dendrobine was reported to have analgesic, antipyretic, hypotensive, and hypothermic activities and to produce moderate hyperglycemia, as well as solve barbiturate poisoning, but little is known about its anti-influenza activity.26 At present, the anti-influenza virus activity of dendrobine in vitro was assessed and its antiviral mechanism was explored.

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MATERIALS AND METHODS

Chemicals. Dendrobine was purchased from Mansite Biotechnology Co. with 98% purity (Chengdu, China), and ribavirin and zanamivir were from Sigma-Aldrich (St. Louis, MO, USA). CL385319, having a purity of > 98%, was synthesized in our laboratory, and its MS (EI) m/z, 1H NMR, and 13C NMR spectra are presented in Figure S1 of the Supporting Information. Cells and Viruses. Madin Darby canine kidney (MDCK) and 293T (human embryonic kidney) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. A549 (human lung epithelial) cells were grown in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin. Influenza A virus subtypes including A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 H274Y (H1N1), and A/Aichi/2/68 (H3N2) were multiplied in 10-day-old chick embryo at 37 °C, and then allantoic fluid containing the above virus was stored at −80 °C and quantified by plaque assay until required. Plasmids. HIV backbone plasmid (pNL4-3.luc.R-E-), A/Thailand/ Kan353/2004-HA, and A/Thailand/Kan353/2004-NA were kindly supplied by Professor Frank Kirchhoff (University Ulm, BW, Germany). pHW2K-NP, pHW2K-PA, pHW2K-PB1, pHW2K-PB2, and pPolI-Fluc (firefly luciferase reporter plasmid) were kindly supplied by Professor Bojian Zheng (University of Hong Kong, Hong Kong, China). A Renilla luciferase plasmid (hRluc-TK) was purchased from Promega (Madison, WI, USA). Cytotoxicity Assay. The MTT assay was used to evaluate the cytotoxicity of dendrobine.27 Briefly, approximately 90% confluent cells in 96-well plates were exposed to dendrobine at 2-fold serial dilutions. After 48 h of incubation, 100 μL of MTT solution, which was diluted by the medium to 0.5 mg/mL, was added and retained at 37 °C for 4 h. Subsequently, the supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan product. The absorbance for each well was measured at 570 nm using the Tecan Genios Pro microplate reader (Bedford, MA, USA). The half-maximal cytotoxic concentration (CC50) was calculated using CalcuSyn software. 3666

DOI: 10.1021/acs.jafc.7b00276 J. Agric. Food Chem. 2017, 65, 3665−3674

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Journal of Agricultural and Food Chemistry a Ti-Eclipse inverted fluorescence microscope (Nikon, Tokyo, Japan) for the 24 h pi. Time-of-Addition Assay. Two kinds of time-of-addition studies were chosen to investigate the period of the viral life cycle interfered with by dendrobine with minor modifications.32,33 For pre-infection, the influenza virus was incubated with dendrobine at 37 °C for 30 min prior to infection and then the co-incubation was added to the cells for an additional 1 h. After incubation, the mixture of unbound viruses and dendrobine was removed. Cells were continuously cultured in fresh medium. For post-infection, dendrobine was added at 1 h after the virus infection as “antiviral assay” as described. For entire infection, the cells were infected as the pre-infection described. After removal of the mixture of unbound viruses and dendrobine, the infected MDCK cells were cultured in fresh medium containing dendrobine at 37 °C for 48 h. The antiviral activity of dendrobine against the virus in three methods of addition experiments was determined by the MTT assay as described above. In another experiment, we further investigated which stage of viral life cycle was disturbed by dendrobine as previously reported.33 In brief, the infected MDCK cells were treated with 50 μg/mL dendrobine at indicated time intervals (0−2, 2−4, 4−6, 6−8, and 0−8 h), which cover the first viral replication cycle. The infection was arrested during the first viral replication cycle. At 8 h pi, the expression of protein was analyzed by Western blotting as mentioned above. Pseudovirus Neutralization Assay. H5N1 pseudovirus was prepared with membrane proteins of NA and HA from influenza A/ Thailand/Kan353/2004 (H5N1) and capsid protein from HIV as Liu et al. described previously.34 In brief, 2 μg of A/Thailand/Kan353/ 2004-HA, 2 μg of A/Thailand/Kan353/2004-NA, and 3 μg of HIV backbone plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E-) were transfected into 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The supernatants containing influenza pseudovirus were harvested at 48 h pi. The pseudovirus titers were quantitated via using luciferase substrate (Promega). VSV-G pseudotyped particles, as negative control, were produced in the same way using a plasmid encoding VSV envelope glycoprotein to replace H5N1 envelope plasmids. For the neutralization assay, H5N1 or VSV-G pseudoviruses were incubated with serially diluted dendrobine or CL-385319 (a positive control) at 37 °C for 30 min before being added to MDCK cells for an additional 48 h. Subsequently, supernatants were discarded, and the cell lysates were transferred to 96-well white luminometer plates prior to the addition of luciferase substrate into the plates. The luciferase activity was measured using an Ultra 384 luminometer (GENiosPro, TECAN, Bedford, MA, USA). Neuraminidase Inhibition Assay. The influence of dendrobine on the release of viral particles was evaluated by NA inhibition assay. Briefly, as described,35 15 μL of influenza virus A/Aichi/2/68 (H3N2) solution was mixed with 5 μL of 2-fold diluted dendrobine or zanamivir (a positive control) in a 96-well black plate at 37 °C for 30 min. Then, 30 μL of 20 μM MU-NANA (2-(4-methylumbelliferyl)-αD-N-acetylneuraminic acid sodium, Sigma-Aldrich) substrate solution dissolved in diluted buffer (32.5 mM MES and 4 mM CaCl2, pH 6.5) was added to each well. The plate was further incubated at 37 °C for 1 h in the dark, followed by adding 50 μL of 14 mM NaOH to end the enzyme reaction. Fluorescence intensity of the product 4-methylumbelliferone was recorded at the excitation wavelength of 340 nm and emission wavelength of 440 nm using a microplate reader. The inhibition rate of NA activity was calculated by using the following formula:

respectively. After incubation for 48 h, the IC50 of each combination against the virus was tested by using the MTT assay as described above. Finally, the fractional inhibitory concentration index (FICI) was calculated using the following formula: FICI = [(IC50of the compound in combination)/(IC50of the compound alone)] + [(IC50of zanamivir in combination)/(IC50of zanamivir alone)]

FICI < 0.5 was interpreted as a significant synergistic antiviral effect. Mini-replicon Assay. The effect of dendrobine on the activity of the viral ribonucleoprotein complex (vRNP) was determined by using a mini-genome assay as previously described.36 Briefly, 293T cells grown in 24-well plates were transfected with 50 ng of NP and viral polymerase plasmids (pHW2K-NP, pHW2K-PA, pHW2K-PB1, pHW2K-PB2) and pPolI-Fluc (a firefly luciferase reporter plasmid) together with 10 ng of hRluc-TK (Renilla luciferase plasmid) using Lipofectamine 2000. At 5 h after transfection, the supernatant was displaced by fresh DMEM with 10% FBS containing various concentrations of dendrobine, zanamivir, or 0.1% DMSO. At 24 h post-transfection, cells were lysed by cell lysates (Promega) for 20 min, and luciferase activity was measured as mentioned above. Surface Plasmon Resonance (SPR) Analysis. The binding affinity of the influenza NP and dendrobine was detected by the PlexArray HT system (Plexera Bioscience, Beijing, China) according to our previous paper with minor modifications.19 After dendrobine was immobilized on a chip surface using photo-cross-linking, recombinant influenza NP at 2-fold serial dilutions was injected at a flow rate of 2 μL/s with a contact time of 300 s and a dissociation time of 300 s. The running buffer is 10 mM phosphate buffer with 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20. The chip platform was regenerated with glycine−HCl (pH 2.0) and washed with the running buffer. The affinity or KD value was calculated with PlexeraDE software by curve fitting using the Langmuir equation. Docking Method. The binding of dendrobine with influenza A/ WSN/1933 (H1N1) NP protein, which was derived from the RCSB protein data bank (PDB: 2IQH), was performed using AutoDock software.33 Before molecular docking, the protein and dendrobine were assigned AMBER ff14SB force field and AM1-BCC charges, respectively, and then 1000 different conformations were generated. The electrostatic interactions and van der Waals forces between dendrobine and the binding sites of NP were acquired to calculate Grid scores. Then the optimal conformation was obtained by cluster analysis (RMSD threshold of 2 Å). Finally, Chimera software was used to analyze simulated results and PyMOL software to generate docking images. The conservation of dendrobine binding sites in swine, avian, and human influenza A viruses was analyzed using Python script. Statistical Analysis. All statistical analyses of the data were performed using GraphPad Prism. The results are expressed as the mean ± standard deviation (SD) from experiments in triplicate. Statistical significance between two groups was analyzed by Student’s t test, more groups by one-way ANOVA with or without Tukey− Kramer multiple comparison. A p value of 99% conservation in >30,000 NP sequences. We wondered whether vRNP activity would change if tested with the site-directed mutant of these active site residues in NP. As shown in Figure 6B, these mutants including R267A, V270A, F338A, and E339A dramatically attenuated vRNP activity, whereas S402A,37,38 located at the homo-oligomerization domain of NP but not essential for vRNP activity, had no such outcome, implying that R267, V270, F338, and E339 are the functional sites of NP and affect viral replication. According to previous research, the NP region (aa 256−340) can bind to the PB2 polymerse subunit of IAV and then become involved in viral RNA replication;39,40 the region (aa 248−274) is functional NES3-NP that can assist the cytoplasmic localization

Table 2. Antiviral Effect of Combination Treatment of Dendrobine and Zanamivir combination ratio (IC50)

IC50 equivalenta

dendrobine:zanamivir

dendrobine

zanamivir

FICIb

10:1 5:1 1:1 1:5 1:10

0.60 0.39 0.33 0.09 0.63

0.06 0.08 0.33 0.45 0.06

0.66 0.47c 0.66 0.54 0.69

a

IC50 equivalent was calculated by dividing the IC50 of drug in combination with its IC50 alone. bFICI was the sum of dendrobine and zanamivir IC50 equivalent in each combination. cFICI < 0.5 was interpreted as significant synergistic effect.

model and identify the essential amino acid (aa) residues for the binding of dendrobine to NP. On the basis of the 3D molecular structure of dendrobine and the monocrystal NP protein structure from PDB, we conducted docking studies and obtained five potential conformations (data not shown) by using AutoDock molecular modeling simulation software. Then studying aa residues in NP that were responsible for the 3670

DOI: 10.1021/acs.jafc.7b00276 J. Agric. Food Chem. 2017, 65, 3665−3674

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Figure 6. Interaction between dendrobine and NP. (A) Molecular docking was performed to create a potential model and identify the essential amino acid residues for the binding of dendrobine to NP. Chimera software was used to analyze simulated results and PyMOL software to generate docking images. (B) vRNP activity of NP mutants in 293T cells. 293T cells were transfected with plasmids pPolI-Fluc, hRluc-TK, pHW2K-PB1, pHW2K-PB2, pHW2K-PA, and pHW2K-NP plasmids or -NP mutant. NP (WT) was used as positive control. Data represent the average of three independent experiments and are shown as the mean ± SD (∗∗∗, p < 0.001).

Table 3. Conservation of Dendrobine Binding Sites in Human, Avian, and Swine Influenza A Viruses position

amino acid residue

267 270 338 339

R V F E

mutant population (conservation ratio %)a 11 184 21 11

(99.97) (99.49) (99.94) (99.97)

a

The 30,000 NP sequences derived from human, avian, and swine influenza A viruses were obtained from NCBI Influenza Virus Resource. The Python script was used to analyze their conservation.

export of the NP via R267 and V270, break NP oligomerization via E339, and interfere with the binding of NP to PB2 subunits via F338.

Figure 5. Identification of NP as the potential target of dendrobine. (A) Affinity between dendrobine and NP. Dendrobine was immobilized on a sensor chip. Subsequently, recombinant influenza NP at 2-fold serial dilutions was injected as analytes. The affinity constant KD is the ratio of the dissociation constant Kd with the association constant Ka, KD = Kd/Ka. (B) Effect of dendrobine on the nuclear export of NP. MDCK cells were inoculated with influenza A/ Aichi/2/68 (H3N2) virus at an MOI of 1.0 and treated with dendrobine or zanamivir for 6 h. The cytoplasmic localization of NP was observed using a confocal laser scanning microscope with anti-NP specific monoclonal antibody and Alexa 488-conjugated goat antimouse secondary antibody (green). The nuclei were counterstained with DAPI (blue). Original magnification, 60×.

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DISCUSSION Alkaloid is one of the active chemical components in Traditional Chinese Medicine and has quite extensive pharmacological effects. Dendrobine, the main alkaloid obtained from D. nobile, possesses a picrotoxane skeleton, and its biological activity is similar to that of picrotoxin.43 Previous studies primarily focus on cardiovascular application of dendrobine.44,45 Here, we found dendrobine had potent antiviral activity against influenza viruses, which had not yet been reported. The results suggested that dendrobine could inhibit the infection of different influenza virus strains on MDCK and A549 cells in a concentration-dependent manner (Figure 1 and Figure S2). Further experiments demonstrated

of NP.41 The salt bridge formed by E339 and R416 is involved in NP oligomerization.42 Therefore, dendrobine may inhibit the 3671

DOI: 10.1021/acs.jafc.7b00276 J. Agric. Food Chem. 2017, 65, 3665−3674

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Journal of Agricultural and Food Chemistry the productions of viral HA mRNA, HA, and NP proteins were remarkably inhibited by dendrobine (Figure 2 and Figure S3). These above results impelled us to investigate the anti-influenza mechanism of dendrobine. The influenza virus can accomplish one life cycle within 8− 10 h.32 To our knowledge, the whole life cycle of the influenza virus could be divided into three stages: entry, replication, and release. HA and NA are enveloped proteins of the influenza virus that mediate viral entry and release. We found dendrobine could not disturb viral entry into cells and the release of progeny viruses via H5N1 pseudovirus neutralization assay and NA inhibition assay, respectively (Figure S4). However, dendrobine effectively inhibited influenza virus infection when cells were treated with dendrobine in post-IAV infection (Figure 3A). These experiments mean dendrobine may target other proteins closely related with virus replication. Subsequently, the result from time of treatment during the first viral replication cycle indicated dendrobine performed an antiinfluenza effect on the early stage of viral replication (0−4 h pi) (Figure 3B,C). Additionally, dendrobine and zanamivir had a synergistic antiviral effect on viral infection (Table 2). Taken together, these results suggest that the targets of dendrobine may be the viral proteins involved in an early stage of viral replication. The vRNP is known as a complex playing an irreplaceable role in viral RNA transcription and replication.46 We explored the influence of dendrobine on vRNP activity. Interestingly, the result showed that dendrobine could lead to a dose-dependent decrease in luciferase activity, which depended on luciferase expression catalyzed by vRNP in the 293T cells (Figure 4A). Moreover, the syntheses of NP vRNA at 3 and 6 h pi were reduced by denrobine (Figure 4B). According to this, the mechanism of dendrobine against influenza A virus infection might be involved in the vRNP complex. The vRNP complex consists of NP, PB1, PB2, and PA. The three subunits of viral polymerase regulate the transcription of viral mRNA through its unique “cap-snatching” mechanism and complete the replication of viral RNA.47 To demonstrate this hypothesis of antiviral mechanism, we first showed that dendrobine could not target the isolated functional domains of the influenza RNA polymerase. Then, we investigated whether dendrobine had an impact on the function of NP. NP is an important component of vRNP, participating in the nuclear translocation of vRNP, the replication of viral RNA, and the assembly and maturation of virus particles.48 On the basis of the biochemical and structural properties of NP that have been published, it has been proposed to be a magnetic target for the development of anti-influenza drugs. Among the functional domains of NP, three novel nuclear export signals (NES1, NES2, NES3) are responsible for mediating the nuclear export of vRNP complex. NES3 is CRM1 dependent ,and the nuclear export of NP can be blocked by leptomycin B (LMB). NES1 and NES2 are CRM1 independent.41 NP also has a tailloop binding pocket that mediates NP−NP interaction. It has been shown in previous research that NP homo-oligomer coupled to the vRNA is likely responsible for the formation of double-helical viral RNP structure, which is related to vRNP functions including viral replication, nuclear export, and genome packaging.49 Furthermore, the E339−R416 salt bridge has a significant effect on NP oligomerization and viral survival. Although mutant E339A and R416A could constitute a heterocomplex with NP, such a complex is incapable of binding the RNA polymerase, resulting in a failure of viral replication.42

In addition, NP possesses an NP-PB2 binding domain, which is localized in the body region of NP and crucial for viral RNA replication.39 With the wealth of knowledge from these studies, identification of small-molecule inhibitors that specifically disrupt the NP function has emerged as an innovative and promising approach. In the current study, we found that dendrobine could interact with NP (Figure 5A). Moreover, the result from observing NP localization with confocal laser scanning microscopy revealed dendrobine inhibited the export of NP from the nucleus to the cytoplasm and decreased the expression of NP protein dependently upon the dose (Figure 5B). We speculated that dendrobine may perform the inhibitory activity via inhibiting the function of NP protein. To further validate the effect of dendrobine on NP, molecular docking was performed to create a virtual binding of dendrobine to NP. The result demonstrated that dendrobine could bind to NP via R267, V270, F338, and E339 with salt bridges, hydrophobic interactions, or hydrogen bonds (Figure 6A). R267 and V270 are located at NP-NES3 (aa 248−274), and F338 is located at NP-PB2 binding domain (aa 256−340), whereas the E339 salt bridge is important to NP oligomerization. Further studies indicated these four amino acids are the conservative functional sites of NP using Python script and the mutants of NP significantly reduced vRNP activity (Table 3 and Figure 6B). This may explain why dendrobine could inhibit the nuclear export of NP, decrease vRNP activity, and then damage viral RNA replication. Unfortunately, the role of F338 at the NP-PB2 binding domain was unclear. Therefore, further studies to illustrate the interference of denrobine in NP-PB2 binding and to verify the function of amino acid residues are underway in our laboratory. To summarize, our present study demonstrated that dendrobine could interfere with the nuclear export of NP and its oligomerization by binding to the critical residues in the functional region of NP, leading to the inhibition of viral replication. Our study provides a fresh perspective for the strategic development of a safe, effective, and affordable natural product for treating influenza virus infection. Importantly, the antiviral activity of dendrobine will become a new application and provide a reference for the full use of D. nobile.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00276. MS(EI) m/z, 1H NMR, and 13C NMR spectra of CL385319; effect of dendrobine on cell viability, influenza A virus replication in A549 cells; H5N1 and VSV-G pseudovirus infection, as well as NA activity; primer sequences for quantitative real-time PCR; positive and negative controls in SPR (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(S.L.) Phone: +8620-6164-8590. E-mail: [email protected]. *(J.Y.) Phone: +8620-6164-8590. Fax: +8620-6164-8655. Email: [email protected]. ORCID

Shuwen Liu: 0000-0001-6346-5006 Jie Yang: 0000-0003-1789-690X 3672

DOI: 10.1021/acs.jafc.7b00276 J. Agric. Food Chem. 2017, 65, 3665−3674

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This work was supported by the Natural Science Foundation of Guangdong Province (No. 2016A030313591), the National Natural Science Foundation of China (U1301224), and the Pearl River S&T Nova program of Guangzhou (No. 2014J2200033). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Frank Kirchhoff for plasmids and Professor Bojian Zheng and members of the Zheng laboratory for reagents and technical help.

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DOI: 10.1021/acs.jafc.7b00276 J. Agric. Food Chem. 2017, 65, 3665−3674