key: cord-0752529-6uuitwip
authors: Kwon, Hyung-Jun; Jeong, Jae-Ho; Lee, Seung Woong; Ryu, Young Bae; Jeong, Hyung Jae; Jung, Kyungsook; Lim, Jae Sung; Cho, Kyoung-Oh; Lee, Woo Song; Rho, Mun-Chual; Park, Su-Jin
title: In vitro anti-reovirus activity of kuraridin isolated from Sophora flavescens against viral replication and hemagglutination
date: 2015-08-31
journal: Journal of Pharmacological Sciences
DOI: 10.1016/j.jphs.2015.04.007
sha: 5b5b47da22809b8c85f26dbfe9f4e7444354ed05
doc_id: 752529
cord_uid: 6uuitwip

Abstract In this study, we evaluated the anti-reovirus activity of kuraridin isolated from the roots of Sophora flavescens. In particular, we focused on whether this property is attributable to direct inhibition of reovirus attachment and/or inhibition of viral replication with the aid of time-of-addition (pre-treatment, simultaneous treatment, and post-treatment) experiments. No significant antiviral activity of kuraridin was detected in the pre-treatment assay. In the simultaneous assay, the 50% effective inhibitory concentrations (EC50) of kuraridin were 15.3–176.9 μM against human type 1–3 reoviruses (HRV1–3) and Korean porcine reovirus (PRV). Kuraridin completely blocked binding of viral sigma 1 protein to sialic acids at concentrations lower than 82.5 μM in the hemagglutination inhibition assay. Moreover, kuraridin inhibited HRV1–3 and PRV viral replication with EC50 values of 14.0–62.0 μM. Quantitative real-time PCR analysis disclosed strong suppression of reovirus RNA synthesis at the late stage (18 h) of virus replication by kuraridin. The viral yields of kuraridin-treated cells were significantly reduced at 24 h post-infection, compared with DMSO-treated cells. Our results collectively suggest that kuraridin inhibits virus adsorption and replication by inhibiting hemagglutination, viral RNA and protein synthesis and virus shedding, supporting its utility as a viable candidate antiviral drug against reoviruses.

Mammalian reovirus (MRV) is the prototype member of the Reoviridae family of non-enveloped double-stranded RNA (dsRNA) viruses, with a genome composed of ten segments. Reoviruses, originally referred to as "respiratory enteric orphans", were first isolated from humans in the United States and Mexico in the 1950s (1) . MRVs are represented by four prototype strains: type 1 Lang (T1L), type 2 Jones (T2J), type 3 Dearing (T3D) and type 4 Ndelle (T4N), with the majority of strains assigned to serogroups T1L, T2J or T3D (2) . Reoviruses have been isolated from the respiratory and enteric tracts of children with mild respiratory or gastrointestinal symptoms (1) . However, limited studies have focused on human reovirus-associated neurological disease to date (3) . Pneumonia and other respiratory diseases in both naturally and experimentally infected primates (4) , and isolation of reoviruses from children with meningitis and encephalitis are widely documented (5, 6) .

MRVs infect a wide range of hosts, including livestock (pig, cattle and chickens), contributing to severe economic losses worldwide. The use of antiviral agents is not commonplace because of their toxicity and high production costs. Hence, effective and inexpensive alternatives to antiviral drugs remain an urgent unmet medical need (7) . Many traditional medicinal plants display strong antiviral activities, and their utility in the successful treatment of infected animals and humans has been demonstrated (8, 9) .

The life cycle of viruses is divided into several stages, including cell surface attachment, penetration, uncoating, replication (protein synthesis) and release, all of which provide targets for antiviral agents (9) . Currently, more than 40 antiviral drugs are licensed for the treatment of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpesviruses. However, the number of licensed antiviral drugs for treatment of highly pathogenic RNA virus infections remains limited (10) . These viral diseases are considered difficult to treat with selective antiviral chemotherapy, highlighting the need for further refinement of antiviral drug design and development. Medicinal plants have a variety of chemical constituents, including alkaloids, tannins, saponins, flavonoids, terpenoids, lignans and coumarins, known to inhibit the replication cycles of various DNA and RNA virus types. Compounds derived from natural sources are therefore of significant interest as possible sources of viral infection control (7e9).

Dried roots of Sophora flavescens Ait. (S. flavescens) have been historically used in traditional Chinese herbal medicine, owing their anti-inflammatory, antiarrhythmic, antipyretic, antiasthmatic, and antiulcerative effects, and for the treatment of diarrhea, gastrointestinal hemorrhage, and eczema (11) . Additionally, a formulation containing S. flavescens is reported to inhibit angiogenesis in a collagen-induced arthritis rat model (12) . In previous studies, quinolizidine alkaloids, flavonoids, and triterpenoids were isolated from the roots of S. flavescens (13) . Recently, quinolizidine alkaloids and flavonoids have been shown to exhibit a wide spectrum of pharmacological activities, including anticancer, antiinflammatory, antitumor, cardioprotective, neuroprotective, antibacterial, and anti-influenza properties (11,13e21) . However, the potential anti-reovirus activities of extracts and compounds isolated from S. flavescens have not been examined to date. In the current investigation, we evaluated the abilities of the MeOH extract, EtOAc fraction and kuraridin isolated from S. flavescens to inhibit human type 1e3 reoviruses (HRV1e3) and Korean porcine reovirus (PRV). Antiviral assays were employed to determine whether the S. flavescens compounds alter reovirus activity by inhibiting virus attachment and/or replication.

The 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were obtained on JEOL ECS400 spectrometer, with CD 3 OD as a solvent. The ESI-MS was determined using an Agilent 6430 LC/MS/MS and 1100 LC/MS spectrometer. Reversed-phase CC was carried out using RP-C18 silica gel (Cosmosil 140C18-PREP, 140 mm, Nacalai tesque, INC.). Silica gel CC was conducted using Kieselgel 60 (70e230 and 230e400 mesh, Merck). TLC was conducted using Kieselgel 60 F254 plates (Merck).

The S. flavescens were purchased at an herbal market in Jeongeup, Korea. A voucher specimen (PB-012e012) has been deposited in the Korea Plant Extract Bank, Korea Research Institute of Bioscience and Biotechnology. Dried roots of S. flavescens (5 kg) were extracted with MeOH (10 L) for 7 days at room temperature. The MeOH extract was evaporated in vacuo, yielding a residue (193 g). The residue was suspended in distilled water (1 L) and extracted with n-hexane, CHCl 3 , EtOAc and BuOH. In this process, nhexane (3.9 g), CHCl 3 (22 g), EtOAc (33.5 g) and BuOH (38.9 g) layers were obtained respectively. The EtOAc layer was submitted to open column chromatography on silica gel (230e400 mesh, 300 g, 

Fetal rhesus monkey kidney (TF-104) cells were grown in Eagle's minimum essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS) and 100 U/mL penicillin, and 100 mg/mL streptomycin and 100 U/mL amphotericin B. Cells were maintained at 37 C with 5% CO 2 . Antibiotic, trypsin-EDTA, FBS, and EMEM were supplied by Gibco BRL (Grand Island, NY, USA). The reovirus Type 1 (Lang, ATCC VR-230), Type 2 (Jones, ATCC VR-231), Type 3 (Dearing, ATCC VR-824) purchased from American Type Culture Collection (ATCC) and PRV (KRP113 strain) isolated from fecal samples of Korean diarrheic piglets were used in this study. Reoviruses were preactivated with 10 mg/mL trypsin (GIBCO Invitrogen Corporation, CA, USA) for 30 min at 37 C before being inoculated onto confluent TF-104 cells and infected cells were maintained in the presence of 1 mg/mL trypsin (GIBCO Invitrogen Corporation, CA, USA).

TF-104 cells were grown in 96 well plates at 1 Â 10 5 cells/well for 48 h. The media in plates were replaced with media containing serial diluted the MeOH extract, EtOAc fraction (2.3e350 mg/mL) or kuraridin (2.3e350 mM) and incubated for 24 h or 72 h. The solution was replaced with only media and 5 mL MTT (3e(4,5edimethyl thiazole2eyl)e2,5ediphenyltetrazolium bromide, SIGMA) solution was added to each well and incubated at 37 C for 4 h. After removal of supernatant, 100 mL 0.04 M HCl-isopropanol was added for solubilization of formazan crystals. Absorbance was measured at 540 nm with subtraction of the background measurement at 655 nm using a microplate reader. Cell viability was calculated as a percentage of the total number of 0.5% DMSO-treated control cells. The CC 50 was calculated as described (23).

The antiviral assays have been previously described (24) , and the visualization of these assays was performed by neutral red method as briefly described.

Pre-treatment assay ( Fig. 2A) : TF-104 cells were grown in 96 well plates at 1 Â 10 5 cells/well for 48 h. Before virus inoculation, non-cytotoxic concentration ( CC 50 ) of the MeOH extract and EtOAc fraction or kuraridin isolated from S. flavescens were added to the cells and incubated for 12 h. Then extracts and compound were removed and the TF-104 cells were washed 2 times with PBS. Reoviruses at a multiplicity of infection (MOI) of 0.01 were inoculated onto the TF-104 cells for 1 h with occasional rocking. The media was removed and replaced by EMEM containing 1 mg/mL trypsin. The cultures were incubated for 72 h at 37 C under 5% CO 2 atmosphere until the cells in the infected, untreated control well showed complete viral cytopathic effect (CPE) as observed by light microscopy. Each concentration of extracts and compounds was assayed in triplicate.

After 72 h incubation in all antiviral assays, 0.034% neutral red was added to each well and incubated for 2 h at 37 C in the dark. The neutral red solution was removed and the cells were washed with PBS (pH 7.4). Destaining solution (containing 1% glacial acetic acid, 49% H 2 O, and 50% ethanol) was added to each well. The plates were incubated in the dark for 15 min at room temperature. Absorbance was read at 540 nm using a microplate reader. The EC 50 that is defined as the concentration offering 50% inhibition of viral yield in cells was calculated as described (23) .

Simultaneous treatment assay (Fig. 2B) : Various concentrations of MeOH extract and EtOAc fraction or kuraridin were mixed with virus at 0.01 MOI and incubated at 4 C for 1 h. The mixture were inoculated onto near confluent TF-104 cell monolayers (1 Â 10 5 cells/well) for 1 h with occasional rocking. The solution was removed and the media was replaced. The cultures were incubated for 72 h at 37 C under 5% CO 2 atmosphere until the cells in the infected, untreated control well showed complete viral CPE as observed by light microscopy. Each concentration of extracts or compound was assayed in triplicate. Table 1 Antiviral activities of the MeOH extract, EtOAc fraction, and kuraridin isolated from S.

flavescens against reovirus for the simultaneous-and post-treatment assay.

Extract or compound Post treatment assay (Fig. 2C ): Reoviruses at 0.01 MOI were inoculated onto near confluent TF-104 cell monolayers (1 Â 10 5 cells/well) for 1 h with occasional rocking. The media was removed and replaced by EMEM with MeOH extract and EtOAc fraction or kuraridin at different concentration. The cultures were incubated for 72 h at 37 C under 5% CO 2 atmosphere until the cells in the infected, untreated control well showed complete viral CPE as observed by light microscopy. Each concentration of extracts and compound was assayed for virus inhibition in triplicate.

The hemagglutination inhibition assay was performed to evaluate the effects of the MeOH extract and EtOAc fraction or kuraridin on viral adsorption to target cells. The reoviruses solution (4 HAU/ 25 mL) was mixed with an equal volume of the extracts or compound (25 mL) in a two-fold serial dilution in PBS (pH 7.4) for 1 h at 4 C. The prepared solution 50 mL was mixed with an equal volume of 1% human red blood cells (hRBC, type O) in HRV1 or 1% bovine red blood cell (bRBC) in HRV2-3 and PRV suspension and incubated for 1 h at room temperature (25, 26) . (27) . The total RNA was reverse transcribed into cDNA using the High Capacity RNA-to-cDNA master mix (Applied Biosystems) according to the manufacturer's instruction. Reverse transcription was performed at 42 C for 1 h. The enzyme was inactivated at 95 C for 5 min. The cDNA was stored at À20 C or directly used in quantitative real-time PCR. Real-time PCR was conducted using 2 mL of cDNA and Power SYBR Green PCR 2X master mix (Applied Biosystems). Cycling conditions for real-time PCR were as follows: 95 C for 1 min, followed by 40 cycles of 95 C for 15 s and 60 C for 15 s. Real-time PCR was conducted using the Step One Plus Realtime PCR system, and the data were analyzed with StepOne software v2.1 (Applied Biosystems). 

The TF-104 cells were infected with HRV1e3 and PRV strain at 0.01 MOI in 6-well plates. After 1 h of virus adsorption at 37 C, the cells were washed three times with PBS and cultured in a medium containing with MeOH extract and EtOAc fraction (20e150 mg/mL) or kuraridin (6.25e50 mМ) at different concentration. The untreated cell and virus controls (0.5% DMSO) were included. The supernatants were harvested after 24 h. The virus yields were determined using plaque assay for 7 days. All determinations were performed thrice in triplicate.

All experiments were repeated at least three times. The differences between groups were assessed using one-way or two-way ANOVA, followed by Tuckey post-hoc analysis. Data were presented as mean ± ANOVA combined standard errors of the mean (S.E.M). A values of P < 0.05 were considered to be significant as compared to the untreated control.

The cell viability of TF-104 cultures treated with the two extracts and kuraridin was evaluated using the MTT assay (Fig. 3) . CC 50 (50% cytotoxic concentration) values for the MeOH extract and EtOAc fraction were 253.3 and 278.4 mg/mL, respectively, while that for kuraridin was considerably lower at 302.2 mM (Table 1) .

Accordingly, all experiments evaluating antiviral effects were performed at concentrations of minimal toxicity below 150 mg/mL for the MeOH extract and EtOAc fraction or 150 mM for kuraridin.

A pre-treatment assay was performed to examine the inhibitory effect of the two extracts and kuraridin on reovirus attachment into host cells (Fig. 2A) . The simultaneous treatment assay was additionally conducted to determine whether the extracts and kuraridin directly inhibit reovirus particles (Fig. 2B) . In the pre-treatment assay, the MeOH extract, EtOAc fraction and kuraridin showed no inhibitory effects against HRV1e3 and PRV (data not shown). Notably, however, in the simultaneous treatment assay, two extracts inhibited reovirus entry (Fig. 4A) . The MeOH extract and EtOAc fraction exhibited a decreasing order of antiviral activity against HRV3, PRV, HRV1, and HRV2, while kuraridin exhibited antiviral activity in a decreasing order against PRV, HRV3, HRV1, and HRV2 (Table 1) . Interestingly, our data indicate that both the extracts and kuraridin exert stronger inhibitory effects on HRV3 and PRV than HRV1 or HRV2, with the greatest inhibitory effect of kuraridin against PRV isolated from Korean porcine diarrheic feces.

The simultaneous treatment assay established that either virus adsorption or cell entry is inhibited by the two extracts and kuraridin. Accordingly, we evaluated whether the extracts and kuraridin inhibit reovirus-induced hemagglutination binding of HRV1 to hRBC or HRV2e3 and PRV to bRBC. The MeOH extract and EtOAc fraction completely inhibited HRV1 attachment to hRBCs as well as HRV2e3 and PRV attachment to bRBCs at concentrations less than 92.5 mg/mL (Fig. 5) , with a decreasing order of HI activity as follows:

PRV, HRV3, HRV2, and HRV1 for the MeOH extract, and PRV, HRV3, HRV1, and HRV2 for the EtOAc fraction. Kuraridin exhibited a similar decreasing order of HI activity against PRV, HRV3, HRV1, and HRV2. Overall, the MeOH extract, EtOAc fraction, and kuraridin showed stronger HI activity against HRV3 and PRV than HRV1 or HRV2, consistent with the findings of the simultaneous assay. Our results clearly indicate that strong interactions of the two extracts and kuraridin with hemagglutinin on the outer-layer protein (sigma 1) of reovirus result in inhibition of viral attachment.

The post-treatment assay was performed to evaluate the inhibitory effects of the MeOH extract, EtOAc fraction, and kuraridin on reovirus replication (Fig. 2C) . The MeOH extract and EtOAc fraction inhibited viral replication in preliminary experiments (Fig. 4B) , with a decreasing order of activity against HRV3, PRV, HRV2 and HRV1 (Table 1) . Kuraridin exerted decreasing antiviral activity in the following order: PRV, HRV3, HRV2, and HRV1 (Table 1 ). Similar to the results of the simultaneous treatment assay, all three isolate fractions showed stronger inhibitory effects on HRV3 and PRV than HRV1 or HRV2. Viral RNA levels synthesized at the early and late stages of virus infection were compared between kuraridin-treated in the number of fluorescent cells infected with PRV (Fig. 7C) . To determine the effects of the two extracts and kuraridin on reovirus production, we performed the virus yield reduction assay. Viral yields were estimated using a plaque assay. Notably, the MeOH extract, EtOAc fraction, and kuraridin suppressed production of HRV1e3 and PRV in a dose-dependent manner indicating inhibition of reovirus shedding or release (Fig. 8 ).

Various steps of the viral replication cycle are targets of antiviral agents, including adsorption, cell penetration, uncoating, transcription, translation, assembly and viral release from infected cells (4) . We hypothesized that the antiviral effects of S. flavescens can be divided into two steps: 1) blockage of virus adsorption to cells and/ or 2) inhibition of viral replication after cell entry. Time-of-addition experiments were performed to determine the stage at which inhibitory activities are exerted. MeOH extract, EtOAc fraction, and kuraridin of S. flavescens were added to TF-104 cells at three distinct time-points, specifically, prior to infection (pre-treatment), at the same time as virus infection (simultaneous) or post-infection (posttreatment) (Fig. 2) .

Reovirus entry into cells is a multistep process involving several interactions between its outer layer protein (sigma 1 protein) and cell surface receptors, including sialic acid (SA) and junctional adhesion molecule-A (JAM-A) (28) . The sigma 1 protein specifies tissue tropism and is responsible for hemagglutination (HA) activity and binding of SA (29, 30) . The HA activity of reoviruses strongly implies a role of SA in cell binding and infectivity of reoviruses, similar to influenza A virus, rotavirus, various coronaviruses and Sendai virus (31) . In the current study, the HI assay was employed to assess the inhibitory effects of the extracts and kuraridin on viral adsorption to host cells. The MeOH extract and EtOAc fraction completely inhibited HRV1 adsorption to hRBCs as well as HRV2e3 and PRV adsorption to bRBCs at concentrations below 92.5 mg/mL. Kuraridin induced complete inhibition of hemagglutination activity of HRV1 with hRBCs and HRV2e3 and PRV with bRBCs at concentrations below 82.5 mM (Fig. 5) . I21nterestingly, the HI activity of kuraridin was stronger for HRV3 and PRV than HRV1 and HRV2. These differences are linked to sigma 1 protein sequences and possibly attributable to serotypespecific interactions of viral proteins with different cell surface receptors. Five residues (Asn198, Arg202, Leu203, Pro204, and Gly205) in sigma 1 protein are reported to play a role in reovirus and SA interactions (32) . Additionally, SA binding by sigma 1 protein of type 3 reovirus is mediated by an eight-stranded cross b-sheet of only the T(iii) domain (residues 175e234) and hemagglutination by type 1 reovirus mediated by the T(iii) and T(iv) domains (residues 248e314) (33) . Thus, the diversity of sigma 1 protein according to type 1e3 Fig. 8 . Reduction of reovirus production by the MeOH extract, EtOAc fraction, and kuraridin. TF-104 cells were infected with HRV1e3 and PRV at MOI of 0.01, and treated with DMSO (0.5%), MeOH extract (20e150 mg/mL) (A), EtOAc fraction (20e150 mg/mL) (B) or kuraridin (6.25e50 mM) (C). After 24 h, supernatant fractions were harvested and virus titers determined with the plaque assay. Data are expressed as mean ± ANOVA combined standard error of three independent assays. *P < 0.05 compared to DMSO-treated cells that were used as control.

serotypes may influence interactions of kuraridin with hemagglutinin on the protein. HI assay results were in agreement with those of the simultaneous treatment assay (Table 1) , indicating that the extracts and kuraridin potentially exert anti-reoviral activity via blockage of viral attachment to SA at the host cell surface. Inhibition of virus attachment, in turn, prevents virus entry, replication and occurrence of infection.

The extracts and kuraridin significantly inhibited reovirus replication after infection in a dose-dependent manner in the posttreatment assay (Fig. 4B ). Although the mechanisms by which kuraridin inhibits reovirus replication are currently unclear, the compound is known to affect viral factors, such as RNA and protein. Our experiments showed that viral RNA levels are significantly lowered by kuraridin. Interestingly, reoviral RNA synthesis was significantly inhibited by kuraridin at the late stage (18 h) of the viral replication cycle for HRV3 and PRV (Fig. 6A) . The reovirus replication cycle comprises two distinct transcription phases (primary and secondary), which occur at early and late stages, respectively. For primary transcription, viral RNA rapidly replicates at 6e8 h post-infection within progeny viral particles. Secondary transcription is mediated by particles assembled from the newly synthesized RNA and protein molecules, and mature virions are produced at >12 h post-infection (4). Our findings indicate that kuraridin inhibits viral protein synthesis (sigma 1) to produce the same changes in viral RNA levels and reduces virus shedding (Figs. 7 and 8). Based on these results, we suggest that the kuraridin suppresses reovirus replication via inhibition of viral RNA, protein synthesis and virus release.

Ribavirin is reported to exert anti-reovirus activity, inhibiting viral multiplication (34) . A number of natural products have been extensively investigated for their antiviral and virucidal activities (35e39). Gallate derivatives that display antiviral effects are used as antioxidant food additives, including EÀ310 (propyl gallate), EÀ311 (octyl gallate), and EÀ312 (lauryl gallate) (39) . Mycophenolic acid has additionally been identified as an anti-reoviral agent, acting as a reversible inhibitor of eukaryotic IMP dehydrogenase (IMPDH) (40) . Another study demonstrated that chestnut and quebracho wood extracts containing tannin show antiviral activity against avian reovirus and metapneumovirus (41) . The dipeptide, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-FA-FMK), is a novel potent inhibitor of reovirus pathogenesis and oncolysis in vivo (42) . These drugs act by inhibiting reovirus replication and adsorption, but their potential side-effects are yet to be clinically evaluated. These natural products may be ideal candidates, since they are less toxic, more effective, have fewer side-effects, and are less expensive than commercially available anti-reovirus agents.

The results from the current study collectively indicate that compounds isolated from S. flavescens may be superior to anti-reovirus agents, and further highlight the medical importance of identifying effective natural antiviral agents.

None.

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This research was supported by a grant from the KRIBB Research Initiative Program, Republic of Korea.