Journal of Virology & Antiviral ResearchISSN: 2324-8955

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Research Article, J Virol Antivir Res Vol: 4 Issue: 2

Cordycepin an Adenosine Analogue Executes Anti Rotaviral Effect by Stimulating Induction of Type I Interferon

Shampa Deb Chanda1, Anindyajit Banerjee2, Satabdi Nandi1, Saikat Chakrabarti2 and Mamta Chawla Sarkar1*
1Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata-700010, West Bengal, India
2Structural Biology and Bioinformatics Division, Council for Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, WB 700032, India
Corresponding author : Mamta Chawla Sarkar
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata-700010, West Bengal, India
Tel: +91-33-2353-74706749
E-mail: [email protected]
Received: April 01, 2015 Accepted: May 19, 2015 Published: May 25, 2015
Citation: Chanda SD, Banerjee A, Nandi S, Chakrabarti S, Sarkar MC (2015) Cordycepin an Adenosine Analogue Executes Anti Rotaviral Effect by Stimulating Induction of Type I Interferon . J Virol Antivir Res 4:2. doi:10.4172/2324-8955.1000138

Abstract

Abstract

Objectives:

Rotavirus is the single most common etiological pathogen of severe diarrhea in infants causing death of over half a million infants a year. In spite of such a huge disease burden, still no effective antivirals are available against rotavirus. Cordycepin (3’-deoxyadenosine), an adenosine analogue, has been reported to modulate cell proliferation, platelet aggregation and provide protection against HIV infection. Herein, attempts were made to analyze efficacy of cordycepin for protection against rotavirus and underlying mechanism.

Methods

Cordycepin’s anti-rotaviral activity was elucidated against simian rotavirus strain SA11 (H96), rhesus strain RRV, human strain Wa (in vitro) and murine strain EW (in BALB/c). Plaque assay, qPCR for viral transcripts and immunoblotting of viral proteins were done to examine viral abundance. Effect of cordycepin on Interferon pathway was analyzed by immunoblotting, co-immunoprecipitation and molecular docking.

Results

Cordycepin was found to reduce propagation of different rotavirus strains such as SA11, RRV, Wa at 64μM with minimal cytotoxicity in vitro and EW in BALB/c mice. Protection was obtained upto 24 hours, after virus infection at both low and high multiplicity of infection. Sensitivity of rotavirus infection to Interferons has been documented. In cordycepin treated cells increased induction of Interferon and downstream antiviral proteins were observed. Cordycepin treatment leads to RIGI-MAVS interaction, IRF3 activation, and Interferon induction. By using different software probable docking sites and orientation of cordycepin and RIG-I were predicted. Cordycepin was found to have relatively higher binding probability for ATP binding domain than other domains. In Vero cells,deficient in Interferon signalling, cordycepin failed to protect against rotavirus. This suggests that though cordycepin may have other effects but anti-rotaviral effects were due to boosting of cellular innate immune responses mediated by Interferon.

Conclusions

Overall this study highlights that cordycepin exerts its anti-rotaviral activity by modulating immune signalling through enhanced Interferon production.

Keywords: Rotavirus; Cordycepin; BALB/c; Interferon I; IRF3; RIG-I-MAVS; Molecular docking

Keywords

Rotavirus; Cordycepin; BALB/c; Interferon I; IRF3; RIG-I-MAVS; Molecular docking

Introduction

Rotavirus (RV), a nonenveloped virus belonging to family Reoviridae, is considered as the most common cause of severe and dehydrating diarrhea in children [1]. Due to lack of proper disease management and unavailability of health care facilities RV accounts for half a million deaths among infants annually [2]. Majority of these deaths occur in lower socioeconomic countries because of limited access to health care facilities as well as rotavirus vaccines, RotaTeq (Merck and Co., PA, USA) and Rotarix (GSK Biologicals, Rixensart, Belgium) [3]. Other than vaccination and Oral rehydration solution for prevention of dehydration, no other effective antivirals have been successfully implemented for treatment of RV infection. Early studies have reported that pretreatment of cells with Interferon (IFN) can restrict RV replication effectively [4]. Supportingly, IFN treatment of newborn calves and piglets prior to RV infection reduces virus replication and disease severity [5-7]. Both high and low sensitivities of bovine RV infection in response to bovine IFN-α/β have been reported from cell culture [8,9]. Thus, the virus is sensitive to the IFN induced antiviral effects.
Cordycepin (3’-deoxyadenosine) is a derivative of nucleoside adenosine, where hydroxyl group at the 3’ position of ribose sugar is replaced by hydrogen. Cordycepin is naturally found in parasitic fungus Cordyceps miltaris [10]. It has been shown to modulate numerous cellular functions such as induction of apoptosis [11-13], inhibition of cell proliferation [14-16] and inhibition of platelet aggregation [17]. It has also been shown to prevent polyadenylation of mRNA [18] and provide protection against human immunodeficiency virus infection [19]. Based on its biological activities, cordycepin is currently being investigated and developed by the National Cancer Institute in the USA as an anticancer drug [20].
In this present study we have analyzed the role of cordycepin in inhibiting RV infection. Cordycepin mediates its anti-rotaviral effect by stimulating the host induced Type I IFN response in cells. RV can efficiently block host induced IFN responses through Nonstructural Protein 1 (NSP1) for efficient virus infection. RV NSP1 binds IRF3, 5 and 7 and mediates their degradation by the proteasome [21-23]. But cordycepin mediated IFN induction create an effective antiviral milieu to block RV replication. This is the first report showing that cordycepin can activate cytoplasmic RIG-I and can induce Type I IFN through RIG-I-MAVS-pIRF3 axis.

Materials and Methods

Ethics statement
The animal investigation was approved by the Institutional Animal Ethics Committee, National Institute of Cholera & Enteric Diseases, Indian Council of Medical Research (Registration No.- NICED/CPCSEA/AW/(215)/2012-IAEC/ SSO) and (Approval # 65/20/08/2009), registered under “Committee for the Purpose of Control and Supervision of Experiments on Laboratory Animals ”, Ministry of Environment and Forests, Government of India and conforms with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health Publication 85–23, revised 1996.
Viruses, cells and viral infections: The monkey kidney cells (MA104), human embryonic kidney 293T cells and human colorectal adenocarcinoma HT29 cells were grown at 37°C in 5% CO2 atmosphere in minimal essential medium (MEM) (MA104), Dulbecco modified Eagle medium (DMEM) (HT29, 293T) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 2 mM sodium pyruvate and 1X PSF (penicillin, streptomycin and fungizone). The simian RV strain SA11 (H96), rhesus strain RRV, human strain Wa, murine strain EW were used for this study. For infection, viruses were activated with acetylated trypsin (10 ug/ml) at 37°C for 30 minutes (min). Confluent monolayers of cells were washed with phosphate buffer saline (PBS) and pre activated viruses were added at 2 multiplicity of infection (2 M.O.I.), unless differently specified. Cells were inoculated with pre activated viruses for 60 min at 37°C. After absorption period the viral inoculums were removed and cell monolayers were washed with PBS to remove unbound viruses. Infection was continued in fresh MEM or DMEM in the absence of serum and trypsin 1X unless differently specified. For multi-step virus growth assay, infected cells were incubated in the medium containing 1 μg/ml trypsin-1X. For all experiments time of virus removal was taken as 0 hour post infection (hpi). Extracted and purified viral preparations were titrated by plaque assay [24]. During measurement of viral titer through plaque assay in presence of drug, cordycepin was added at 64 μM with agarose overlay containing DMEM, trypsin. For negative control 0.1% DMSO was added in place of cordycepin. Plates were incubated at 36°C in a humidified atmosphere of 5% CO2 in air. After 12 hours (h) and 24 h plaques were stained overnight with 0.1% neutral red and plaques were counted. This experiment was performed in triplicate.
M.O.I. was calculated from plaque forming unit (pfu) by the following formula: Multiplicity of infection (M.O.I.)=Plaque forming units (pfu) of virus used for infection / number of cells.
Rotavirus infection mouse model
BALB/c mice were raised in filter-topped cages on a standard rodent diet with water available ad libitum. The experiments were performed according to national regulations and approved by the concerned animal ethics committee. Four-day-old mice were inoculated with EW RV strain (107 PFU) through oral gavages. After 12 h of virus inoculation, mice were treated with either cordycepin (0.5-10 mg/kg/day) or equivalent concentration of DMSO solution for 5 days by oral gavages. Faecal samples were collected from each mouse at 1 to 5 days post infection (dpi), and presence of virus in the stool was detected by Premier®Rotaclone® (Meridian Bioscience, Inc) as per manufacturer’s protocol. Total RNA was isolated from small intestinal homogenate isolated from sacrificed mice at 5 dpi followed by real time analysis with RV NSP4.
Reagents, antibodies and treatments: Cordycepin (3’Deoxyadenosine) was purchased from Sigma-Aldrich, St. Louis, MO, USA, and dissolved in dimethyl sulfoxide (DMSO) for all studies. Rabbit polyclonal antibodies against SA11 NSP1 (480–497 amino acids) and NSP3 (full length) and NSP4 peptide fragment were gifted by Professor Taniguchi, Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan. Antibodies against phospho-Janus kinase 1 (p-JAK1) (3331S), Janus kinase 1 (JAK1) (3332S), p-IRF3 (Ser396) (4947S), IRF3 (4302S), p-Signal Transducer and Activator of Transcription 1 (p-STAT1) (9171S), STAT1 (9172S), GAPDH (2118S), Retinoic Acid Inducible gene I (RIG-I) (3743S), Oct1 (8157S), were from Cell Signaling Inc., Danvers, MA, USA. Antibody against FLAG epitope (SAB4200071) was from Sigma. Antibodies against His probe (sc-803) and IRF-7 (sc-9083) were from SantaCruz Biotechnology (CA, USA). Antibody against Mitochondrial antiviral-signaling protein MAVS (06-1096) was purchased from Millipore (Billerica, MA). All antibodies were used at manufacture recommended dilution. For anti NSP4, NSP1 and NSP3 antibody 1:3000 dilution was used.
Cytotoxicity assay: MultiTox-Glo Multiplex Cytotoxicity Assay kit (Promega) was used to measure cytotoxicity of cordycepin. In brief HT29 and 293T cells were seeded in 96 well culture plates. At about 70%-80% confluency, cordycepin was added at concentrations ranging from 0.5-256 μM. 48 h after drug induction cytotoxicity of cordycepin was assessed by measuring live cell fluorescence at ~400nmEX/ ~505nmEMas per manufacturer’s protocol. Assays were performed in triplicate to confirm the results.
Nucleus Isolation: Confluent monolayers of HT29 cells were either mock infected or infected with RV (2 M.O.I) and incubated with cordycepin (64 μM). At different time intervals (3 and 8 hpi), nuclear extracts were prepared with a ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas Life Science) for IRF7 translocation studies.
Immunoblot analysis: Whole cell lysates [extracted with Totex buffer (20 mM Hepes at pH 7.9, 0.35 M NaCl, 20% glycerol, 1% NP- 40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 50 mM NaF and 0.3 mM Na3VO4) containing mixture of protease and phosphatase inhibitors (Sigma, St. Louis, MO)], cytoplasmic or nuclear extracts or immunoprecipitated products were prepared. Samples were incubated in protein sample buffer (final concentration: 50 mM Tris, pH 6.8, 1% SDS, 10% glycerol, 1% β-mercaptoethanol, and 0.01% bromphenol blue), boiled for 5 min and subjected to SDS-PAGE followed by immunoblotting according to standard protocols [25] using specific primary antibodies. Primary antibodies were identified with HRP conjugated secondary antibody (Pierce, Rockford, IL) and chemiluminescent substrate (Millipore, Billerica, MA). To confirm protein loading blots were reprobed with GAPDH or Oct1. All immunoblots shown are representative of three independent experiments.
Coimmunoprecipitation: Infected or transfected cells were washed with cold PBS and then lysates were clarified by incubation with protein A-Sepharose beads (2h) at 4°C followed by centrifugation. Supernatant was incubated with anti-FLAG and anti-MAVS antibodies overnight at 4°C, followed by incubation with protein A-Sepharose for 4h. Beads were washed 5 times with 1ml wash buffer (200 mM Tris pH-8.0, 100 mM NaCl and 0.5% NP-40) and bound proteins were eluted with SDS sample buffer. Eluted protein samples then separated on 12% SDS-PAGE gels followed by immunoblotting with anti-RIG-I or anti-MAVS and anti-FLAG antibodies.
Quantitative Real Time PCR: Total RNA was isolated using TRIzol (Invitrogen, Grand Island, USA) following manufacturer’s instructions. cDNA was prepared from 2 μg of RNA using the Superscript II reverse transcriptase (Invitrogen) with random hexamer primers. Real-time PCR reactions (50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s and 72°C for 10 min) were performed in triplicate using SYBR Green (Applied Biosystems, Foster City, CA, USA). Primers specific for vp6 (F-5′-CAGTGATTCTCAGGCCGAATA- 3′; R-5′-GGCGAGTACAGACTCA CAAA-3′), gapdh (F-5’-GTCAACGGATTTGGTCGTATTG-3’ and R-5’-TGGAAGATGGTGATGGGATTT-3’), nsp4 (F-5’-GGATCCAGGAATGGCGTATTT- 3’ and R-5’-TTCTAGCTGGCGTCTCATTTC- 3’), nsp3 (F-5’-AACAGATGGCCGTCTCAATTA-3’ and R-5’-GAAGTATCAGCAAGCCAGTTTC-3’), nsp1(F-5’GCCTAACTACGTGGCATCTAAT- 3’ and R-5’-ACACTGGCGACATGTGATT- 3’), ifn-α (F-5′-CTGGCACAGATGAGGAGAAT-3’ and R-5′-CTGCTGGTAGAGTTCAGTGTAG-3’), ifn-β (F-5′-GCTCCAGAACATCTTTGCTATTT- 3’ and R-5′- TCCTTGGCCTTCAGGTAATG- 3’), ifn-γ (F-5’-GTGGAGACCATCAAGGAAGAC-3’ and R-5’-,ACCTCGAAACAGCATCTGAC-3’) rig-I (F-5’-GACATGGGACGAAGCAGTATT- 3’ and R-5’-GGGATGTGGTCTACTCACAAAG- 3’) and mx1 (F-5’- GAAGATAAGTGGAGAGGCAAGG-3’ and R-5’-.GGTTATGCCAGGAAGGTCTATT-3’) were used for corresponding gene transcripts. The relative gene expressions were normalized to gapdh using the formula 2-ΔΔCT (ΔΔCT=ΔCT Sample-ΔCTUntreated control).
Luciferase reporter assay: IRF3 luciferase reporter gene assays were performed by transfecting pTATA-luc-4x-IRF3 plasmid in 293T cells with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instruction. pTATA-luc-4x-IRF3 contains the luciferase reporter gene under the control of four copies of IRF- 3 binding PRDI/III motif of the IFN-β promoter [26]. For NFKB lucifecrase reporter assay 293T cells were cotransfected with NFKBluc (containing IL8 promoter) and pRL-TK. 24 h post transfection, cells were infected with SA11 strain and subsequently incubated with either cordycepin or Poly-IC. At 3 hpi and 10 hpi, the luciferase activity was measured according to the manufacturer’s protocol (Promega) using a luminometer (Varioskan multimode reader, Thermo Fisher). The relative luciferase activity of NFKB-luciferase was normalized with Renilla luciferase. The experiment was repeated three times to confirm the results.
Plasmid and siRNA, shRNA Transfection: Vector pEF-BOSRIG- I, expressing wt human RIG-I and pEF-BOS-RIG-KA270 expressing mutant RIG-I (mutation in Walker ATPase motif) are generous gifts from Dr. Takashi Fujita (Kyoto University). Full length NSP1 was cloned in pcDNA6 vector (Invitrogen) with C-terminal His-Tag as described previously [27]. Custom-synthesized siRNA against RIG-I was obtained from Dharmacon. Plasmids or siRNA were transfected in 293T cells with Lipofectamine 2000 (Invitrogen) or siPORT-NeoFX (Ambion) respectively according to manufacturer’s instructions.
For generating stable MAVS knockdown cell lines, a MAVS shRNA Plasmid (sc-75756-SH from SantaCruz Biotechnology, CA, USA) was transfected into 293T cells as per manufacturer’s instructions. Stably transfected cells were selected in media containing puromycin (10 μg/ml; Sigma), and individual cell clones were isolated and confirmed by immunoblotting.
Bioinformatical analysis: Crystal structure of retinoic acidinducible gene 1 (RIG-I) (protein data bank ID: 3TMI) [28] from Homo sapiens was retrieved from the protein data bank (PDB) [28,29]. Structural cavity analysis was performed using the CASTp web server [30]. Three-dimensional (3D) coordinates of cordycepin was collected from the PubChem database [31]. Molecular docking of cordycepin onto the RIG-I structure was performed using the GOLD v5.2 [32], PatchDock [33] and FireDock [34] programs. PatchDock provided rigid-body protein-protein docking solutions whereas FireDock aims to refine each docking solution by restricted interface side-chain rearrangement and by soft rigid-body optimization. For this analysis default parameters were used for PatchDock and FireDock runs. GOLD software optimizes the fitness score of many possible docking solutions using a genetic algorithm. Following parameters were used in the GOLD docking cycles: population size -100, selection pressure -1.100000, number of operations -100,000 number of islands -5, niche size -2, and crossover weight 95, mutate weight -95, and migrate weight -10. 100 docking calculations were run for the ligand and the best docking solution was identified based on critical manual inspection satisfying favorable interactions between the ligand and the protein molecule. GOLD score, PatchDock score and, FireDock scores were further converted to Z score statistics to identify the best docking solutions. Chemscore function in GOLD package estimates the total free energy change that occurs on binding of the ligand and is represented as ΔG of binding [35,36].ΔGbinding= ΔG0 + ΔGhbondShbond + ΔGmetalSmetal + ΔGlipoSlipo + ΔGrotHrot.
Where Shbond, Smetal, and Slipo denote scores for hydrogen bonding, acceptor-metal, and lipophilic interactions, respectively. Hrot represents the loss of conformational entropy of the ligand upon binding to the protein; the ΔG terms are coefficients derived from a multiple linear regression analysis on a training set known of protein–ligand complexes [35].
A protein-ligand clash-energy term, Eclash is also used to differentiate ligand-protein binding solutions. It is summed over all non-hydrogen protein–ligand atom pairs and it is calculated using following formula:
Eclash =∑ εclash(r, rclash)
Where r is the distance between a protein ligand atom pair and rclash is the clash distance for that pair. The clash energy (εclash) for each atom pair depends on the nature of the protein and ligand atom.
Satistical Analysis: Data are expressed as mean ± S.D. of at least three independent experiments (n≥3). In all tests, P=0.05 was considered statistically significant.

Results

Anti-rotaviral activity of cordycepin in cell lines (in-vitro) and in mice model (in vivo)
Cytotoxicity assay was performed on HT29 and 293T cells with increasing concentration of cordycepin (0.5 μM-256 μM) by MultiTox Multiplex Cytotoxicity Assay kit (PROMEGA). In brief, HT29 cells, treated with different concentrations of cordycepin (0.5 μM-256 μM), were incubated with 2X MultiTox-Fluor Multiplex Cytotoxicity Assay Reagent. Live cell fluorescence was measured at ~400nmEX/ ~505nmEM as described in Material and methods. As shown in Supplementary Figure 1A, compared to untreated control cells, 50% cell death was observed at 256 μM (LD50). Similar results were obtained in 293T cells (Supplementary Figure 1B). Thus in subsequent experiments, nontoxic doses of cordycepin (8 μM to 64 μM) were used. The anti-rotaviral activity of cordycepin was evaluated in vitro in SA11 (H96) infected HT29 cells. Confluent monolayers of cells were mock-infected or infected with SA11 RV strain (2 M.O.I.) for 1h at 37°C and treated with different concentrations of cordycepin after virus adsorption. At 12 hpi a marked reduction in the transcript level of nsp4 mRNA was observed in presence of cordycepin (64 μM) (Figure 1A left panel). Similarly reduction in NSP4 protein level was also observed in cordycepin treated infected cells compared to mock treated infected cells (Figure 1A right panel). Relative expression pattern of different nonstructural proteins (nsp1 and nsp4) was also assessed. Cells were infected with RV in presence of 64 μM cordycepin and total RNA was isolated followed by real time analysis in a time course dependent manner. During early hours (1 hpi) no significant differences in nsp transcripts were observed in presence of cordycepin whereas, during later hours (>5 hpi) significant reduction of viral transcripts was observed (Figure 1B) suggesting that probably viral entry in cells is not affected by cordycepin. Consistent with these results, at 12 hpi and 24 hpi a significant reduction in viral titers was observed in cordycepin treated SA11 infected cells compared to mock treated SA11 infected cells (Figure 1C). Cordycepin mediated antiviral activity was independent of RV strains from human or animal origin as there were a significant reduction in viral titers in both RRV and Wa infected cells, in presence of cordycepin (64 μM) (Figure 1D) .
Figure 1: Antiviral role of cordycepin in rotavirus infected HT29 cells.HT29 cells were infected with rotavirus strain SA11 (2 M.O.I.), after adsorption cells were washed with PBS and incubated with increasing concentrations of cordycepin. (A)Left panel: Following cordycepin treatment total RNA was isolated using TRIZOL at 12 hpi and nsp4 transcripts were analysed by real time PCR. Fold changes were obtained by normalizing relative gene expression to gapdh using formula 2-ΔΔC T (ΔΔCT=ΔCT Sample-ΔCT Untreated control). Right panel: Total cellular extracts from SA11 infected HT29 cells treated with cordycepin (8 μM - 64 μM) were separated by SDS-PAGE to assess expression of viral protein NSP4 by immunoblotting. Results revealed reduced NSP4 expression in 32 and 64 μM cordycepin treated cells. Membrane was reprobed with GAPDH antibody as internal control. (B) SA11 infected HT29 cells were treated with 64 μM cordycepin for 0-24 hpi. RNA was isolated at specified time points and expression of nsp1 and nsp4 transcripts was analysed by real time PCR compared to uninfected control cells. (C) Viral titer was measured by plaque assay after 24 hpi in cordycepin incubated infected cells relative to mock incubated infected cells. (D) HT 29 cells were infected with RRV and Wa strains (2 M.O.I.) followed by cordycepin treatment for 12 hpi and 24 hpi. Virus yield was determined by plaque assay method for indicated time points.Results shown ((A, B, C and D) are obtained as mean of three independent experiments (P < 0.05).
Efficacy of cordycepin to block RV was not dependent on viral titers (M.O.I.) used for infection. SA11 propagation was inhibited under conditions of single step virus growth (in the absence of trypsin, where no reinfection by viral progeny takes place) as well as in multistep virus growth (in presence of trypsin) at both low and high multiplicity of infection (Figure 2A). Furthermore, the efficiency of cordycepin to provide protection against RV was analyzed under three different treatment conditions: Pre treatment, Post treatment, and during RV infection. During Pre treatment, HT29 cells were incubated with cordycepin (64 μM) for 3, 6 or 9 h, washed and then infected with SA11. For Post treatment cordycepin was added at 0 hpi and after 3 hpi. For during adsorption condition cells were incubated with both cordycepin and the virus suspension together; after 1 h of incubation both drug and the virus was removed from the medium. As shown in Figure 2B cordycepin mediated suppression of RV infection was observed both in Pre and Post treatment groups although suppression was more prominent when drug was added after virus adsorption. In Pre treatment condition, protection was not very significant in 9 h pre treated group compared to 3 h and 6 h pre treated groups (Figure 2B). As revealed from Figure 2B, treatment during adsorption had no significant effect on viral titers which further confirmed that cordycepin did not hamper viral entry.
Figure 2: Cordycepin inhibits rotavirus infection independent of M.O.I.(A) HT 29 cells were infected with SA11 at an M.O.I. of 1, 5 and 10 followed by cordycepin treatment for 8 hpi and 24 hpi Single-step (Trypsin was added only during viral adsorption) and multi-step (Trypsin was added during viral adsorption as well as after adsorption). (B) HT 29 cells were incubated with cordycepin at the indicated times before infection (Pre), immediately after adsorption (Post) or only during adsorption. Virus yield was determined by plaque assay method for indicated time points. Data shown is mean of three independent experiments (P < 0.05).
In vivo, BALB/c mice were infected orally with EW RV strain, followed by treatment with increasing concentrations of cordycepin (0.5-10 mg/kg/day) daily for 5 days. Real time analysis revealed reduced RV nsp4 in cordycepin (6.25/mg/kg/day-10mg/kg/day) treated mice compared to mock treated infected mice (Figure 3A). Furthermore, viral titers in stool of cordycepin treated infected mice (5 dpi) were 5-8 folds less compared to mock treated infected mice (Figure 3B). RV was detected in stool samples collected from mice at 5 dpi which confirms viral shedding. In addition decreased accumulation of water in the small intestine of cordycepin (10/ mg/kg/day) treated infected mice was observed compared to mock treated infected mice (Figure 3C).
Figure 3: Anti-rotaviral Activity of cordycepin in mice model.(A), (B)Four-day-old mice were inoculated with EW RV (107PFU) through oral gavages followed by (after 12 h of infection) treated with either increased concentration of cordycepin (0.5-10 mg/kg/day) by oral gavages daily, for 5 days or equivalent concentration of DMSO solution (n=6, n denotes number of mice in each experimental group). RNA was isolated at 5 dpi from mice intestine and expression of nsp4 transcript was analysed by real time PCR compared to mock treated control cell (A). Viral titer from stool sample collected at specified dpi were measured by plaque assay which revealed significant reduction of viral titer in cordycepin treated mice (B). Results shown (A and B) are obtained as mean of three independent experiments (P < 0.05). (C) Part of intestine showing accumulation of water in EW infected mouse compared to cordycepin treated and mock treated mice.
Cordycepin hinders rotavirus infection through induction of IFN
To investigate the mechanism behind anti-rotaviral effects of cordycepin, relative expression of different cytokines including IL1β, IL8, IL10, TNFα, IFN-α, IFN-β, IFN-γ were analyzed (data not shown). Both IFN-α and β were upregulated in cordycepin treated cells irrespective of virus infection or not. These led to further analysis of IFN signalling in response to cordycepin treatment. Confluent monolayers of cells were kept treated with either different concentrations (1-6 4μM) of cordycepin or poly-IC at 10 μg/ml (known as a positive stimulator of IFN ). Total RNA was isolated followed by quantitative PCR for ifn-β after 5h of incubation. Significant increase in transcript was observed in cells treated with 64 μM cordycepin (Figure 4A). Below this concentration measurable induction of ifn-β was not obtained as shown in Figure 4A. ifn-α transcript was also induced in response to cordycepin treatment but no change was observed in ifn-γ expression (Figure 4B).To correlate this finding, IFN induction was studied in cordycepin treated cells in presence of RV infection. ifn-β transcript was analyzed in cells either mock-infected or infected with SA11 strain followed by treatment with cordycepin (64 μM). As shown in Figure 4C, there was a surge in ifn-β transcript (10-16 folds) after 3h and 8 h of cordycepin treatment in both SA11 and mock infected cells. In case of only SA11 infection 1.8 fold induction of ifn-β was observed at 3 hpi which was reduced at 8 hpi (Figure 4C). Consistent to IFN induction, increased phosphorylation of “Janus Kinase 1” (JAK1) and “Signal Transducer and Activator of Transcription 1” (STAT1) as well as induction of IFN stimulated gene mx1 were observed in cordycepin treated SA11 infected or mock infected cells (Figure 4D, 4E). Overall results suggest that cordycepin induces IFN which further activates downstream antiviral genes. Similar experiments were performed in Vero cell line, which is deficient of IFN signaling. Confluent monolayers of cells were either mock-infected or infected with SA11 (2 M.O.I.) for 1h at 37°C and treated with cordycepin. Total RNA was isolated at 6, 12, 18 and 24 hpi and Rotaviral vp6 transcript levels were quantitated in cordycepin treated RV infected cells relative to untreated infected cells. Surprisingly, in Vero cells vp6 transcripts were not low in cordycepin treated cells (Figure 4F left panel), similarly NSP3 protein also revealed no change in expression in presence or absence of cordycepin (Figure 4F right panel).
Figure 4: Cordycepin blocks rotavirus infection through induction of type I IFN. (A) HT29 Cells were treated with increasing concentrations of cordycepin, and an ifn-β mRNA level was analyzed over control cell by real time PCR. Results revealed significant induction of ifn-β transcript at 64 μM of cordycepin. (B) Total RNA was extracted from HT29 cells incubated with cordycepin for 6 hours followed by real time analysis to quantitate expression of ifn-α and γ transcripts, fold changes were obtained by normalizing relative gene expression to gapdh using formula 2-ΔΔC T (ΔΔCT=ΔCT Sample-ΔCT Untreated control). Results reveal induction of only ifn-α.but no change in ifn-γ. transcripts in cordycepin treated cells. (C) HT29 cells infected with SA11 were either left untreated or were treated with cordycepin (64 μM), or DMSO or Poly-IC (10 μg/ml) for 3 hpi and 8 hpi. Total RNA was isolated and ifn-β. transcript level was quantitated using real time PCR, which showed enhanced ifn-β. in cells treated with Poly-IC (positive control) and cordycepin (both uninfected and infected cells) compared to DMSO treated or virus infected cells. (D) and (E) HT29 cells were infected with SA11 (2 M.O.I.) and treated with cordycepin for at indicated time points. Total RNA and whole cell lysates were prepared. Immunoblotting was done to assess phosphorylation of JAK1 and STAT1 proteins which revealed activation of JAK1 and STAT1 proteins in cordycepin treated cells (D). (E). mx1 transcript expression was analysed by real time. Transcript level was induced by 3-4 folds in presence of cordycepin. Fold change in transcript was calculated by normalizing relative gene expression to gapdh using formula 2-ΔΔC T (ΔΔCT=ΔCT Sample-ΔCT Untreated control). (F) Vero cells were infected with SA11 (2 M.O.I.) followed by cordycepin treatment for 6-24 hours. Total RNA and whole cell lysates were prepared and expression of vp6 transcript and NSP3 protein was assessed by real time PCR and immunoblotting respectively. GAPDH gene was used as internal loading control. Results shown are mean of three independent experiments (P < 0.05) at Figure (A, B, C, E and F).
Cordycepin treatment modulates anti IFN activity of rotavirus
During RV infection NSP1 has been shown to suppress IFN induction through multiple pathways including degradation of IRF3, IRF5 and IRF7 [37-39]. To determine whether cordycepin mediated IFN induction can outweigh anti IFN activity of NSP1, expression of IRF3 was analyzed. Confluent mono layers of HT29 cells were infected with SA11 strain (2 M.O.I.). After virus removal cells were either left untreated or were treated with cordycepin (64μM) followed by immunoblotting to check phosphorylation of IRF3 and total IRF3. As shown in Figure 5A, increased phospho IRF3 and total IRF3 was observed in SA11 infected cordycepin treated cells compared to SA11 infected untreated cells. Indeed uninfected cordycepin treated cells showed increased expression of p-IRF3 and total IRF3. To further determine whether increased IRF3 is functional as transcriptional enhancer, activation of IRF3 was analyzed using pTATA-luc-4x- IRF3 vector. In pTATA-luc-4x-IRF 3 vector luciferase reporter gene is controlled by four copies of the IRF3 responsive regulatory (PRD) I/III region of the IFN-β promoter/enhanceosome (4X IRF-3) [26]. In cells treated with cordycepin (both uninfected and virus infected), enhanced luciferase activity was observed compared to SA11 infected untreated cells (Figure 5B). Furthermore in cordycepin treated HT29 cells (both uninfected and SA11 infected cells), IRF7 translocation to nucleus was also observed (Figure 5C). IRF3 after activation dimerizes with IRF7 and translocates to nucleus [40]; so presence of IRF7 in nuclear fraction confirms activation of IRF3. To confirm dominance of cordycepin mediated IFN induction over Rotaviral NSP1 mediated IFN antagonism, 293T cells overexpressing NSP1 were incubated with cordycepin for 10h. Total cell lysates were immunoblotted for IRF3 and increased IRF3 expression was observed in NSP1 overexpressing 293T cells treated with cordycepin compared to NSP1 overexpressing untreated cells (Figure 5D). In identical experimental conditions ifn-β transcripts was also assessed. NSP1 over expressing cells showed enhanced ifn-β transcript in presence of cordycepin (data not shown).
Figure 5: Cordycepin induced Interferon subdues rotavirus mediated inhibition of Interferon (A) and (C) Whole cell lysates were prepared from SA11 infected HT29 cells in presence or absence of cordycepin (64 μM) treatment. (A) Immunoblotting results revealed phosphorylation of IRF3 and restoration of IRF3 in presence of cordycepin. Membranes were reprobed with GAPDH antibody as a loading control. (C) Cytosolic and nuclear proteins were isolated with ProteoJET cytoplasmic and nuclear protein extraction kit. Cytosolic and nuclear fractions were analysed for IRF7, oct1 and GAPDH proteins. (B) IRF3 promoter activity was measured in 293T cells transfected with 4xIRF3 promoter luciferase reporter vector. 24 h post transfection, cells were infected with SA11 followed by treatment with cordycepin (64μM) or Poly-IC (10 μg/ml). At indicated time points, luciferase activities were determined using the LARII reagent and the data are presented as the fold change in luciferase units (mean ± SD, n=3) relative to mock infected control. (D) 293T cells were transfected with pcDNA NSP1 and 24 h post transfection cordycepin (64 μM) was added. Cell lysates were prepared after 10 h followed by immunoblotting with IRF3 antibody. Results indicate restoration of IRF3 protein in NSP1 over expressing cells during cordycepin treatment. Membranes were reprobed with anti-His and anti-GAPDH to confirm over-expression of NSP1 and equal loading respectively.
Cordycepin induces IFN through RIG-I MAVS signalling axis
Since cordycepin was found to induce IFN both in HT29 and 293T cells (data not shown) upstream regulators of IFN were investigated. RIG-I MAVS pathway was analyzed initially as 293T cells have been reported to be deficient in most of the TLR signalling [41]. Effect of cordycepin on both RIG-I protein and transcript level was studied through immunoblot analysis and real time PCR. Confluent monolayers of HT29 cells were infected with SA11 followed by treatment with cordycepin. As a positive control, cells were treated with poly-IC (10 μg/ml). RIG-I expression was checked by immunoblotting at 3 hpi and 8 hpi in whole cell lysates. Results shown in Figure 6A revealed no change in RIG-I expression in virus infected but untreated cells [42] whereas in virus infected; cordycepin treated cells, increased RIG-I expression was observed. A modest but distinguishable increase in RIG-I expression was observed in cordycepin treated uninfected HT29 cells. This might be due to increased IFN secretion in cells. Correspondingly an enhanced transcript level of rig-I was also obtained both in SA11 infected and mock infected HT29 cells after cordycepin treatment for 3h and 8h (Figure 6B). Since RIG-I interacts with MAVS for downstream signaling, aliquots of same cell lysates were immunoprecipitated with anti-MAVS antibody and immunoblotted with anti-RIG-I antibody (Figure 6C). As shown in Figure 6C upper panel, RIG-I immunoprecipitated with MAVS in cells treated with cordycepin (both uninfected and infected) whereas no interaction was observed in SA11 infected; untreated cells. Furthermore, 293T cells were transfected with pEF-BOS-RIG-I plasmid to overexpress RIG-I followed by treatment with cordycepin after 24h. After 8h of cordycepin treatment, cell lysates were prepared and immunoprecipitated with anti-FLAG antibody (for RIG-I) followed by immunoblotting with anti-MAVS antibody. Similar to previous results (Figure 6C) RIG-I immunoprecipitated with MAVS in cells treated with cordycepin (Figure 6D upper panel). Overexpression of RIG-I in cells was confirmed by immunoblotting input cell lysates with anti-FLAG antibody (Figure 6D lower panel). To assess the role of RIG-I in cordycepin mediated IFN induction, 293T cells were transfected with either RIG-I siRNA (20 nM) or nonspecific control siRNA.RIG-I expression was analyzed by immunoblotting after 24 h. Compared to non-specific control siRNA transfected cells, reduced RIG-I expression was observed in cells transfected with RIG-I siRNA (Figure 6E lower panel). In parallel 293T cells, transfected with RIG-I siRNA or control siRNA, were treated with cordycepin for 8 h. IFN induction was assessed by quantitating ifn-β transcript. In cells transfected with RIG-I siRNA, cordycepin failed to induce IFN , whereas ifn-β was induced in control siRNA transfected cells (Figure 6E). Similarly, when induction of IFN was assessed in MAVS-/- 293T cells, reduced IFN induction was observed compared to wt 293T cells treated with cordycepin (Figure 6F).
Figure 6: Cordycepin activates interferon through RIG-I-MAVS signaling pathway.(A) and (B) HT29 cells infected with SA11 (2 M.O.I.) were treated with cordycepin and were harvested at 3 hpi and 12 hpi for real time PCR and western blotting. (A) Immunoblotting confirmed induction of RIG-I protein in presence of cordycepin (64μM). GAPDH was used as an internal loading control. (B) Results show induction of rig-I transcripts in presence of cordycepin in both uninfected and rotavirus infected cells. Simultaneously, HT29 cells were incubated with Poly-IC (10 μg/ml) as a positive control. Fold changes were obtained by normalizing relative gene expression to gapdh using formula 2-ΔΔC T (ΔΔCT=ΔCT Sample-ΔCT Untreated control). Results shown are mean of three independent experiments (P < 0.05). (C) HT29 cells infected with SA11 (2 M.O.I.) were treated with cordycepin (64μM) for 3 hpi and 8 hpi. Whole-cell extracts were immunoprecipitated with anti-MAVS antibody followed by immunoblotting with anti-RIG-I antibody. CO-IP revealed positive interaction between RIG-I and MAVS in cordycepin treated cells. For analyzing expression of proteins in input, cell lysates were immunoblotted with anti-MAVS and anti-RIG-I antibody. (D) 293T cells were transfected with pEF-BOS-RIG-I followed by cordycepin or Poly-IC treatment (10 h). Whole cell lysates were immunoprecipitated with anti-FLAG antibody (for RIG-I). Western blotting with anti- MAVS antibody indicates interaction of FLAG tagged RIG I and MAVS in cordycepin and Poly-IC treated cells. Whole cell lysates were immunobloted with anti- MAVS and anti-FLAG antibody to assess expression of MAVS and RIG-I in input. (E) 293T cells were transfected with siRIG-I and control siRNA (scrambled siRNA) followed by SA11 infection and treatment with either cordycepin or Poly-IC. Total RNA was isolated by TRIZOL to quantitate ifn-β mRNA levels over control cells. Reduction of ifn-β transcripts was observed in RIG-I siRNA treated cordycepin or Poly-IC treated cells compared to control siRNA transfected cells. Results shown are mean of three independent experiments (P < 0.05). Reduced expression of RIG-I in siRIG-I transfected cells was confirmed by immunoblot. (F) Total RNA was isolated from 293T and 293T MAVS-/-cells, infected with SA11 (2 M.O.I.) followed by treatment with cordycepin (64 μM) and expression of ifn-β was measured by real time PCR. Results are representations of mean of three independent experiments (P < 0.05) Reduced MAVS expression in 293T MAVS-/- cells was confirmed by immunoblotting with anti-MAVS antibody. GAPDH was used as an internal loading control.
Once role of RIG-I in cordycepin mediated IFN induction was established, functional role of ATPase activity of RIG-I during induction of IFN was analyzed. RIG-I mutant (K270A) which has a defect in Walker A motif of the helicase-like domain results in no ATPase activity [43]. 293T cells were transfected with RIG-I K270A for 24 h followed by treatment with cordycepin or poly-IC (for 8 h). Whole cell extracts were immunoprecipitated with anti-FLAG antibody (for RIG-I) and immunoblotted with anti-MAVS antibody. As shown in Figure 7E, cordycepin can activate K270A mutant RIG-I, similar to RIG-I, which was assessed by co-immunoprecipitation of MAVS with mutant RIG-I. Similarly in cells treated with poly-IC, RIG-I K270A co-immunoprecipitated with MAVS suggesting that activation of RIG-I signaling for induction of IFN does not require ATPase activity of RIG-I.
Figure 7: Docking analysis of cordycepin with RIG-I structure. (A). Molecular docking programs suggest higher binding probability of cordycepin with respect to ATP binding domain than other domains of RIG-I protein. Docking scores (converted in Z score statistics) obtained from different blind docking programs are displayed in the box plot where the central rectangle spans the first quartile to the third quartile and a segment inside the rectangle shows the median and “whiskers” above and below the box show the locations of the minimum and maximum values. FD_ATP_DM, FD_ATP_DM, PD_ATP_DM, PD_RNA_DM and PD_OTR_DM denote docking results obtained from Patch Dock [27] and Fire Dock [28] when cordycepin is docked at ATP binding domain, RNA binding domain, and others binding domains, respectively. (B). Comparative analysis of ligand binding, represented by ΔG of docking of the cordycepin in absence (-ATP) and presence (+ATP) of ATP with RIG-I structure. Lower the ΔG value higher the probability of stability of binding between the ligand the protein. (C). Comparative analysis of the average GOLD clash score of the cordycepin in absence (-ATP) and presence (+ATP) of ATP with RIG-1 structure. Higher clash score indicates more unfavouredness of the complex is (D). Cartoon representation RIG-I protein structure (PDB ID: 3TMI) where the Rec A-like domain 1 or the ATP binding domain (region: 240-444) is shown in magenta, Rec A-like helicase domain 2 (region: 600-744) in yellow, alpha-helical domain 3 (region: 470-599) in green and the repressor domain (region: 795-923) in grey. The linker (region: 445-469) connecting Rec A-like domain 1 or the ATP binding domain with alpha-helical domain 3 is coloured in blue, while the “V” shape linker (region: 745-794) between Rec A-like helicase domain 2 and repressor domain is coloured in brown. The docked cordycepin is shown in stick representation. Residues within the 5Å of docked cordycepin (PHE 241, LYS 242, PRO 243, ARG 244, TYR 246, GLN 247, VAL 452, VAL 453, GLY 267, CYS 268, GLY 269, LYS 270, THR 271, PHE 272, ARG 732) are highlighted whereas probable hydrogen bonds formed between cordycepin and RIG-I residues are marked by black lines. (E). 293T cells were transfected with pEF-BOS-RIG-I K270A expressing FLAG tagged mutant RIG-I. After 24 h transfected cells were incubated with Cordycepin and Poly-IC. After 10 h of incubation whole cell lysates were prepared, immuno precipitated with anti-FLAG antibody and then precessed for SDSPAGE analysis. Western blotting with anti-MAVS antibody indicate co-precipitating MAVS with FLAG tagged RIG in Cordycepin treated and Poly-IC treated cells in immunoprecipitated fraction and non-immunoprecipitated (INPUT) fractions from the same samples were probed with anti-MAVS (upper panel). Over expression of RIG was confirmed by anti-FLAG antibody (lower panel).
Molecular docking analysis of cordycepin and RIG-I
RIG-I protein structure (PDB ID: 3TMI) consists of four larger domains highlighted in Figure 7D where the RecA-like domain 1 or the ATP binding domain (region: 240-444) is shown in magenta, alpha-helical domain (region: 470-599) in green, RecA-like helicase domain (region: 600-744) in yellow, and the repressor domain (region: 795-923) in grey. The linker (region: 445-469) connecting the ATP binding domain with alpha-helical domain is coloured in blue, while the “V” shape linker (region: 745-794) between RecAlike helicase domain and the repressor domain is coloured in brown. To identify the probable domain for cordycepin binding, was initially docked onto the RIG-I structure using blind or naïve docking approaches employed by PatchDock [31] and FireDock [32] programs, whichsuggests that the cordycepin has relatively higher binding probability for the ATP binding domain than other domains in RIG-I (Figure 7A). Further, a residue specific docking approach employed by the GOLD v5.2 program [30] suggests that the cordycepin binds to RIG-I more efficiently in absence of ATP than in the presence of it (Figure 7B, 7C). Residues that might interact with the cordycepin were identified (Figure 7D) and probable hydrogen bond formation between the cordycepin and residues GLY 267, GLN 247, ARG 244, LYS 242 (Figure 7D) was predicted. Figure 7D also suggested that PHE 241 might be involved in π–π stacking interaction with the adenyl ring of cordycepin to stabilize the interaction.

Discussion

This study identified anti-rotaviral property of cordycepin in vitro and in vivo as measured by both end point viral titer and assessing viral gene expression (Figures 1-3). The anti-rotaviral effects of cordycepin were not restricted to any one RV strain or genotype but it extended to a broad range of RV strains (Figure 1D). Cordycepin treatment both before and after RV infection resulted in significant inhibition of virus, however, no antiviral effects were observed when added during virus adsorption (Figure 2B). This suggested that antiviral activity was not directed towards viral entry step.
To understand the mechanism by which cordycepin may affect cellular signalling, initially virus induced cytokines such as IL1β, IL8, IL10, TNFα, and IFN-α, β, γ were analyzed by real time PCR (data not shown). Among different cytokines, only significant changes in ifn-α and β transcripts were observed, thus IFN signalling was further studied. In both infected and uninfected cells, enhanced transcripts were obtained for both ifn-α and β in presence of cordycepin (Figure 4A, 4B, and 4C). Furthermore activation of JAK-STAT signalling and induction of an ISG (mx1) were also observed in cordycepin treated cells (Figure 4D, E). Previous studies in new born calves and piglets reported treatment with exogenous IFN can effectively suppress RV infection and reduce diseases severity in vitro [4-9]. RV can successfully replicate in cell lines expressing IFN , but their replication is restricted if the cells are initially primed with IFN prior to virus infection [4-6]. It is well documented that RV have evolved various strategies to restrict the expression of type I IFN in infected cells. RV NSP1 has been shown to modulate IFN response by degradation of IRF3, 5, 7 proteins [21-23]. Cordycepin was less potent than poly-IC in inducing IFN , but it was sufficient to induce antiviral status to block RV propagation. Thus cordycepin induces significant amount of Type I IFN which RV NSP1 is not able to antagonize completely in both RV (SA11) infected and in pcDNA-NSP1 overexpressing cells. Cordycepin induced activation of IRF3 and IRF7 were observed (Figure 5A, 5B and 5C) and RV-NSP1 failed to degrade IRF3 induced by cordycepin (Figure 5D). Though cordycepin has been shown to suppress IKB phosphorylation and NFKB translocation to nucleus [44] but anti-rotaviral effects of cordycepin are probably due to its ability to induce IFN as in IFN signalling deficient Vero cells, no antiviral effect of cordycepin was observed (Figure 4F).
RIG-I, a member of RLR family, is a RNA helicase, containing a C-terminal DExD/H box RNA helicase domain as well as two N-terminal caspase activation and recruitment domain (CARDs). RIG-I is an important sensor of RNA virus infection as both single stranded RNA (with 5’-triphosphate) “ssRNA” and double stranded RNA(dsRNA) serve as its ligand resulting in conformational changes and exposure of CARD domain [45]. MAVS, an adaptor protein binds to CARD domain of RIG-I, resulting in dimerization and activation of IRF3-IRF7 complex and subsequent induction of IFN genes [46]. During entry dsRNA of RV remains encapsulated in double layered particle (DLP) within the cytoplasm, thus input dsRNA is not exposed [37]. However during replication ss + RNA transcripts are extruded by DLPs which may engage RIG-I [47]. During RV infection, NSP1 targets RIG-I for degradation and blocks RIG-I-MAVS mediated IFN induction [42]. In this study it was revealed that both in cordycepin treated infected and uninfected cells there was enhanced expression of RIG-I transcripts and protein compared to virus infected cells (Figure 6A, 6B). This might be due to further stimulation of RIG-I expression as an ISG following IFN induction. Furthermore, RIG-I co-immunoprecipitated with MAVS in both cordycepin treated SA11 infected or mock infected cells (Figure 6D, 6E), confirming activation of RIG-I signalling by cordycepin.
After ligand binding, RIG-I requires ATP hydrolysis which is critical for proper antiviral function of RIG-I, but the role for energy utilization in their biological responses is poorly understood [48]. Mutation in Walker A motif that is presumed to be defective in ATPase activity did not affect signaling by at least some dsRNA or poly-IC [43]. Consistent with the previous reports, activation of RIG-I K270A mutant was observed in Poly-IC induction (Figure 7E). Intriguingly, incubation of RIG-I K270A mutant with cordycepin also resulted in activation and further downstream signaling which led to IFN induction (Figure 7E). This suggested that probably cordycepin mediated RIG-I activation is also independent of ATPase activity. Blind docking results clearly revealed that ATP binding domain of RIG-I has better binding affinity with cordycepin than other binding domains (Figure 7A). Furthermore, residue specific docking analysis showed preferred binding of cordycepin with RIG-I in absence of ATP (Figure 7B, 7C). Docking analysis identifies the residues that might be interacting with the cordycepin and further indicates probable hydrogen bond formation between the cordycepin and GLY 267, GLN 247, ARG 244, LYS 242, respectively (Figure 7D). Additionally, PHE 241 might be involved in π–π stacking interaction with the adenyl ring of codycepin to provide stabilization to the interaction.
Overall the study highlights novel IFN inducing property of cordycepin which can be exploited as broad spectrum antiviral for short term acute viral infections (Figure 8). Induction of IFN in cells by cordycepin would overcome the IFN antagonizing mechanism executed by viruses in cells. In addition, cordycepin can also reduce excessive inflammation caused by induction of cytokines like IL8 by blocking IKB phosphorylation and NFKB translocation. It has been shown that cordycepin induces IFN through activation of RIG-I. Further in depth biochemical and in vitro assays are required to fully understand the mechanism behind cordycepin mediated RIG-I activation. However, based on the results cordycepin can be used effectively for treatment of RV in addition to oral rehydration therapy to reduce mortality and morbidity in children.
Figure 8: Putative Model for Mechanism of cordycepin to stimulate Interferon. Cordycepin after entering the cell may serve as a ligand for RIG-I. Following cordycepin binding with RIG-I undergo conformational changes and interact with CARD domain of MAVS protein (residencial protein of mitochondria). RIGMAVS interaction results in IRF3 phosphorylation and nuclear translocation through TRADD-TBK1 signaling molecule. Interferon is enhanced through IRF3/IRF7 phosphorylation and nuclear translocation. Enhanced Interferon creates an antiviral milieu which blocks Rotavirus replication.

Acknowledgements

The study was financially supported by ICMR, India and Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Chanda S. D. is supported by research fellowships from the University Grant Commission (UGC). Banerjee A. is supported from the ‘HOPE’ (BSC-0114) network project of the Council of Scientific and Industrial Research (CSIR) Nandi S. is supported by research fellowship from the Council of Scientific and Industrial Research (CSIR), India. Dr. Chakrabarti S is supported by Ramalingaswami Fellowship, Department of Biotechnology (DBT) and CSIR-Indian Institute of Chemical Biology (IICB).
Antibodies of NSPs were gifted by Professor Koki Taniguchi, Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan. Vectors of wt and mutant human RIG-I are generous gifts from Dr. Takashi Fujita, Kyoto University, Japan.

References

  1. Parashar UD, Hummelman EG, Bresee JS, Miller MA, Glass RI (2003) Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 9: 565-572.

  2. Estes MK. Kapikian AZ (2007) Rotaviruses: Virology. (5thedn). Lippincott-Raven, Philadelphia, USA

  3. Patton JT (2012) Rotavirus diversity and evolution in the post-vaccine world. Discov Med 13: 85-97.

  4. Dagenais L, Pastoret PP, Van den Broecke C, Werenne J (1981) Susceptibility of bovine rotavirus to interferon. Brief report. Arch Virol 70: 377-379.

  5. Lecce JG, Cummins JM, Richards AB (1990) Treatment of rotavirus infection in neonate and weanling pigs using natural human interferon alpha. Mol Biother 2: 211-216.

  6. Schwers A, Vanden Broecke C, Maenhoudt M, Béduin JM, Wérenne J, et al. (1985) Experimental rotavirus diarrhoea in colostrum-deprived newborn calves: assay of treatment by administration of bacterially produced human interferon (Hu-IFN alpha 2). Ann Rech Vet 16: 213–218.

  7. Vanden Broecke C, Schwers A, Dagenais L, Goossens A, Maenhoudt M, et al. (1984) Interferon response in colostrum-deprived newborn calves infected with bovine rotavirus: its possible role in the control of the pathogenicity. Ann Rech Vet 15: 29-34.

  8. Derbyshire JB (1989) The interferon sensitivity of selected porcine viruses. Can J Vet Res 53: 52-55.

  9. La Bonnardiere C, de Vaureix C, L'Haridon R, Scherrer R (1980) Weak susceptibility of rotavirus to bovine interferon in calf kidney cells. Arch Virol 64: 167-170.

  10. cunningham KG, Manson W, Spring FS, Hutchinson SA (1950) Cordycepin, a metabolic product isolated from cultures of Cordyceps militaris (Linn.) Link. Nature 166: 949.

  11. Wu WC, Hsiao JR, Lian YY, Lin CY, Huang BM (2007) The apoptotic effect of cordycepin on human OEC-M1 oral cancer cell line. Cancer Chemother Pharmacol 60: 103-111.

  12. Thomadaki H1, Scorilas A, Tsiapalis CM, Havredaki M (2008) The role of cordycepin in cancer treatment via induction or inhibition of apoptosis: implication of polyadenylation in a cell type specific manner. Cancer Chemother Pharmacol 61: 251-265.

  13. Chen LS1, Stellrecht CM, Gandhi V (2008) RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells. Br J Haematol 140: 682-391.

  14. Chang W, Lim S, Song H, Song BW, Kim HJ, et al. (2008) Cordycepin inhibits vascular smooth muscle cell proliferation. Eur J Pharmacol 597: 64-69.

  15. Shi P1, Huang Z, Tan X, Chen G (2008) Proteomic detection of changes in protein expression induced by cordycepin in human hepatocellular carcinoma BEL-7402 cells. Methods Find Exp Clin Pharmacol 30: 347-353.

  16. Nakamura K, Yoshikawa N, Yamaguchi Y, Kagota S, Shinozuka K, et al. (2006) Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation. Anticancer Res 26: 43–47

  17. Cho HJ1, Cho JY, Rhee MH, Kim HS, Lee HS, et al. (2007) Inhibitory effects of cordycepin (3'-deoxyadenosine), a component of Cordyceps militaris, on human platelet aggregation induced by thapsigargin. J Microbiol Biotechnol 17: 1134-1138.

  18. Lallas GC1, Courtis N, Havredaki M (2004) K562 cell sensitization to 5-fluorouracil- or interferon-alpha-induced apoptosis via cordycepin (3'-deoxyadenosine): fine control of cell apoptosis via poly(A) polymerase upregulation. Int J Biol Markers 19: 58-66.

  19. Müller WE, Weiler BE, Charubala R, Pfleiderer W, Leserman L, et al. (1991) Cordycepin analogues of 2',5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase. Biochemistry 30: 2027–2033.

  20. Kodama EN, McCaffrey RP, Yusa K, Mitsuya H (2000) Antileukemic activity and mechanism of action of cordycepin against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells. Biochem Pharmacol 59: 273–281.

  21. Barro M1, Patton JT (2005) Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci U S A 102: 4114-4119.

  22. Barro M, Patton JT (2007) Rotavirus NSP1 inhibits expression of type I interferon by antagonizing the function of interferon regulatory factors IRF3, IRF5, and IRF7. J Virol 81: 4473-4481.

  23. Graff JW, Mitzel DN, Weisend CM, Flenniken ML, Hardy ME (2002) Interferon regulatory factor 3 is a cellular partner of rotavirus NSP1. J Virol 76: 9545-9550.

  24. Smith EM, Estes MK, Graham DY, Gerba CP (1979) A plaque assay for the simian rotavirus SAII. J Gen Virol 43: 513-519.

  25. Chawla-Sarkar M, Bae SI, Reu FJ, Jacobs BS, Lindner DJ, et al. (2004) Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis. Cell Death Differ 11: 915-923.

  26. Hrincius ER, Dierkes R, Anhlan D, Wixler V, Ludwig S, et al. (2011) Phosphatidylinositol-3-kinase (PI3K) is activated by influenza virus vRNA via the pathogen pattern receptor Rig-I to promote efficient type I interferon production. Cell Microbiol 13: 1907-1919.

  27. Bhowmick R, Halder UC, Chattopadhyay S, Nayak MK, Chawla-Sarkar M (2013) Rotavirus encoded non-structural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53. J Virol 87: 6840–6850.

  28. Jiang F, Ramanathan A, Miller MT, Tang GQ, Gale M Jr, et al. (2011) Structural basis of RNA recognition and activation by innate immune receptor RIG-I. Nature 479: 423-427.

  29. Berman HM1, Westbrook J, Feng Z, Gilliland G, Bhat TN, et al. (2000) The Protein Data Bank. Nucleic Acids Res 28: 235-242.

  30. Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, et al. (2006) CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res 34: W116-118.

  31. Bolton E, Wang Y, Thiessen PA, Bryant SH (2008) Integrated Platform of Small Molecules and Biological Activities: Annual Reports in Computational Chemistry. (4th edn), American Chemical Society, Washington, USA

  32. Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267: 727-748.

  33. Schneidman-Duhovny D1, Inbar Y, Nussinov R, Wolfson HJ (2005) PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res 33: W363-367.

  34. Andrusier N, Nussinov R, Wolfson HJ (2007) FireDock: fast interaction refinement in molecular docking. Proteins 69: 139-159.

  35. Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD (2003) Improved protein-ligand docking using GOLD. Proteins 52: 609-623.

  36. Eldridge MD, Murray CW, Auton TR, Paolini GV, Mee RP (1997) Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes. J Comput Aided Mol Des 11: 425-445.

  37. Arnold MM, Sen A, Greenberg HB, Patton JT (2013) The battle between rotavirus and its host for control of the interferon signaling pathway. PLoS Pathog 9: e1003064.

  38. Sherry B (2009) Rotavirus and reovirus modulation of the interferon response. J Interferon Cytokine Res 29: 559-567.

  39. Hu L, Crawford SE, Hyser JM, Estes MK, Prasad BV (2012) Rotavirus non-structural proteins: structure and function. Curr Opin Virol 2: 380-388.

  40. Paun A, Pitha PM (2007) The IRF family, revisited. Biochimie 89: 744-753.

  41. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdörfer B, et al. (2002) Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168: 4531-4537.

  42. Qin L, Ren L, Zhou Z, Lei X, Chen L, et al. (2011) Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I. Virol J 8: 526.

  43. Ranjith-Kumar CT, Murali A, Dong W, Srisathiyanarayanan D, Vaughan R, et al. (2009) Agonist and antagonist recognition by RIG-I, a cytoplasmic innate immunity receptor. J Biol Chem 284: 1155-1165.

  44. Ren Z, Cui J, Huo Z, Xue J, Cui H, et al. (2012) Cordycepin suppresses TNF-α-induced NF-κB activation by reducing p65 transcriptional activity, inhibiting IκBα phosphorylation, and blocking IKKγ ubiquitination. Int Immunopharmacol 14: 698-703.

  45. Lu C, Xu H, Ranjith-Kumar CT, Brooks MT, Hou TY, et al. (2010) The structural basis of 5' triphosphate double-stranded RNA recognition by RIG-I C-terminal domain. Structure 18: 1032-1043.

  46. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805–820

  47. Trask SD, McDonald SM, Patton JT (2012) Structural insights into the coupling of virion assembly and rotavirus replication. Nat Rev Microbiol 10: 165-177.

  48. Bruns AM, Pollpeter D, Hadizadeh N, Myong S, Marko JF, et al. (2013) ATP hydrolysis enhances RNA recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (LGP2). J Biol Ch em 288: 938-946.

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