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Antiviral activity of lanatoside C against dengue virus infection
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Antiviral activity of lanatoside C against dengue virus infection
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Yan Yi Cheung a, Karen Caiyun Chen a, Huixin Chen a, Eng Khuan Seng b, Justin Jang Hann Chu a,⇑ a Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore b School of Chemical & Life Sciences, 180 Ang Mo Kio Ave 8, Nanyang Polytechnic, Singapore 569830, Singapore
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Article history: Received 18 March 2014 Revised 7 August 2014 Accepted 13 September 2014 Available online xxxx Keywords: Dengue virus Cardiac glycoside Flavivirus Lanatoside C Antiviral compounds
a b s t r a c t Dengue infection poses a serious threat globally due to its recent rapid spread and rise in incidence. Currently, there is no approved vaccine or effective antiviral drug for dengue virus infection. In response to the urgent need for the development of an effective antiviral for dengue virus, the US Drug Collection library was screened in this study to identify compounds with anti-dengue activities. Lanatoside C, an FDA approved cardiac glycoside was identified as a candidate anti-dengue compound. Our data revealed that lanatoside C has an IC50 of 0.19 lM for dengue virus infection in HuH-7 cells. Dose-dependent reduction in dengue viral RNA and viral proteins synthesis were also observed upon treatment with increasing concentrations of lanatoside C. Time of addition study indicated that lanatoside C inhibits the early processes of the dengue virus replication cycle. Furthermore, lanatoside C can effectively inhibit all four serotypes of dengue virus, flavivirus Kunjin, alphavirus Chikungunya and Sindbis virus as well as the human enterovirus 71. These findings suggest that lanatoside C possesses broad spectrum antiviral activity against several groups of positive-sense RNA viruses. Ó 2014 Published by Elsevier B.V.
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1. Introduction
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Dengue virus (DENV), the most significant virus in the Flaviviridae family with the highest morbidity and mortality rates, poses a serious global threat in the past few decades. DENV is serologically subdivided into four serotypes, DENV-1, -2, -3 and -4. The enveloped virus contains a single-stranded, positive-sense RNA genome that is directly translated into a single polyprotein which is eventually cleaved into the ten individual viral proteins. The three structural proteins, envelope (E), capsid (C) and premembrane (prM) proteins form the virion particles (Mukhopadhyay et al., 2005). The remaining seven non-structural proteins are primarily involved in viral RNA replication within the infected cells. Dengue infection can manifest itself either as a mild, self-limiting clinical infection or the more severe dengue hemorrhagic fever (DHF) and even the life-threatening dengue shock syndrome (DSS). Currently, there are numerous candidate compounds (Lim et al., 2013), including the well-known mycophenolic acid and ribavirin (Diamond et al., 2002; Takhampunya et al., 2006) that are reported
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⇑ Corresponding author at: Laboratory of Molecular RNA Virology and Antiviral Strategies, Yong Loo Lin School of Medicine, Department of Microbiology, MD4, Level 5, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore. Tel.: +65 65163278; fax: +65 67766872. E-mail address: [email protected] (J.J.H. Chu).
to possess anti-DENV activities. Nevertheless, none of them are approved yet for the treatment of the disease. Therefore, the development of safe and effective antiviral treatments is now a pressing issue to combat this medically important mosquito-borne viral pathogen. Lanatoside C, a US Food and Drug Administration (FDA) approved cardiac glycoside that acts by inhibiting the Na+-K+-ATPase pump was recently found to inhibit several negative-strand RNA viruses including the influenza virus, vesicular stomatitis virus and Newcastle disease virus (Hoffmann et al., 2008). Another well studied cardiac glycoside, ouabain that acts similarly by inhibiting the Na+-K+-ATPase pump also inhibited herpes simplex virus (Dodson et al., 2007), Sendai virus (Nagai et al., 1972; Tomita and Kuwata, 1978) and murine leukemia virus (Tomita and Kuwata, 1978). Increased intracellular Na+ and reduced intracellular K+ concentrations have both been demonstrated to affect replication of several negative-strand viruses, DNA viruses and retroviruses (Nagai et al., 1972; Hartley et al., 1993; Chen et al., 2004; Hoffmann et al., 2008; Bertol et al., 2011). In our high-throughput screen of the US Drug Collection library for anti-DENV compounds, lanatoside C was identified as a potent inhibitor of dengue virus infection. The inhibitory effect of lanatoside C on DENV-2 was examined by measuring the production of infectious virus particles and viral RNA as well as viral protein expression. The study was also extended to the different DENV
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Please cite this article in press as: Cheung, Y.Y., et al. Antiviral activity of lanatoside C against dengue virus infection. Antiviral Res. (2014), http:// dx.doi.org/10.1016/j.antiviral.2014.09.007
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serotypes and other positive-strand RNA viruses (Kunjin virus, Chikungunya virus, Sindbis virus and Human enterovirus 71). Our data showed dose-dependent inhibition of DENV-2 virus infection, viral RNA production and viral protein synthesis following treatment with increasing concentrations of lanatoside C. Other viruses within the Flaviviridae, Togaviridae and Picornaviridae families were also found to be inhibited by lanatoside C.
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2. Materials and methods
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2.1. Cell lines and viruses
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Human umbilical vein endothelial (HUVEC) cells were purchased from Clonetics, USA. Baby hamster kidney fibroblast (BHK21) cells, human muscle rhabdomyosarcoma (RD) cells and mosquito cell line C6/36 from Aedes albopictus were obtained from American Type Culture Collection (ATCC). The human leukemic monocyte lymphoma (U937) cell line was kindly provided by Professor Ng Mah Lee, Department of Microbiology, National University of Singapore (NUS). The human hepatoma (HuH-7) cell line was a kind gift from Dr. Priscilla Yang, Department of Microbiology and Immunology, Harvard Medical School. HUVEC, BHK21 and U937 cells were maintained in RPMI-1640 medium (Sigma– Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS). HuH-7 and RD cells were maintained in DMEM (Sigma–Aldrich) supplemented with 10% FCS. C6/36 cells were maintained in L-15 medium (Sigma–Aldrich) supplemented with 10% heat inactivated FCS. BHK21, U937, RD and HuH-7 cells were maintained at 37 °C with 5% CO2 in a humidified incubator. C6/36 cells were maintained at 28 °C, in the absence of CO2. DENV serotype 1–4 (Singapore isolates) were grown in C6/36 mosquito cell line derived from A. albopictus. Sindbis virus (SINV) and Kunjin virus (KUNV) were kind gifts from Professor Ng Mah Lee, Department of Microbiology, National University of Singapore. Human Enterovirus 71 (5865/SIN/000009) (HEV71) was kindly provided by A/P Vincent Chow, Department of Microbiology, National University of Singapore and Chikungunya virus (D1225Y08) (CHIKV) was kindly provided Dr. Ng Lee Ching, Environmental Health Institute, National Environment Agency.
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2.2. US drug collection screen
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The US Drug Collection (Microsource, Discovery Systems Inc., Gaylordsville, CT, USA) library consisting of a total of 1040 compounds was screened for compounds that effectively inhibit DENV infection. Briefly, HuH-7 cells were infected with DENV-2 at multiplicity of infection (MOI) of 1 and incubated at 37 °C for 1 h to allow virus adsorption. The cells were then incubated with the different compounds and subsequently processed for immunofluorescence assay as described previously (Low et al., 2011). Inhibition of DENV-2 by the various compounds, relative to the DMSO-treated control was expressed as the percentage of DENV antigen positive cells. The top 20 positive hits displaying greater than 50% inhibition against DENV-2 were selected for further validation of their anti-DENV properties. HuH-7 cells were infected and treated with the selected compounds for validation. The supernatant was collected for quantification of infectious viral titre via plaque assay.
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2.3. Cell viability assay
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AlamarBlueÒ cell viability assay (Invitrogen, Carlsbad, CA, USA) was used to measure the cell viability of the various cell lines following treatment with different concentrations of lanatoside C (Sigma–Aldrich) following manufacturer’s instructions. Sodium azide and 0.1% DMSO were used as experimental controls. The
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fluorescence intensity was measured by the Infinite M200 microplate reader (Tecan, Männerdorf, Switzerland) using excitation and emission wavelengths of 570 nm and 585 nm, respectively. The data was then processed by the i-control 1.6 software (Tecan).
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2.4. Dose-dependent studies
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2.4.1. Infection of cells For DENV-1, DENV-2, DENV-3, DENV-4 and Kunjin virus (KUNV), adherent HuH-7 and HUVEC cells were seeded on 24-well plates at 5 104 and 1 105 cells/well one day prior to infection. Cells were then infected with the different viruses at MOI of 1. For the suspension cell line, U937, cells were seeded at 8 105 cells/well on the day of infection before being infected by DENV-2 (MOI of 1). The cells were then incubated at 37 °C for 1 h to allow virus adsorption. For CHIKV and SINV virus, BHK21 cells were seeded on 24-well plates at 7.5 104 cells/well one day prior to infection. The cells were infected at MOI of 1 as described above for 1 h (SINV) and 1.5 h (CHIKV). For HEV71, RD cells were seeded on 24-well plates at 2 105 cells/well one day prior to infection. The cells were infected at MOI of 1 as described above for 1 h. Following infection, the cells were rinsed with PBS before treatment with lanatoside C.
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2.4.2. Lanatoside C treatment Appropriate volumes of medium (supplemented with 2% FCS) containing various concentrations of drug were added to the infected cells. 0.1% DMSO was used as the vehicle control for comparison with the drug-treated infected cells. The cells were then incubated at 37 °C for either 12 h (SINV and HEV71 infected BHK21 and RD cells), 24 h (CHIKV infected BHK21 cells), 48 h (DENV-1, DENV-2, DENV-3, DENV-4 and KUNV infected HuH-7, HUVEC and U937 cells) before they were harvested for plaque assay.
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2.5. Time-of-addition study
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For the pretreatment assay, HuH-7 cells were treated with lanatoside C for 2 h before infection by DENV-2. For the time-ofaddition study, HuH-7 cells were first infected with DENV-2 at MOI of 1 and lanatoside C was then added at 0, 4, 6, 8, 10, 12, 24 and 48 hpi. The infected cells were processed for immunofluorescence detection at 72 hpi and the percentage of DENV antigen positive cells were quantified.
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2.6. Quantitative RT-PCR
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At 24 and 48 hpi, DENV-2 viral RNA was extracted from the samples, using the RNeasy Mini Kit (QIAGEN), following the manufacturer’s protocols. Briefly, the nucleic acid was first precipitated using 100% ethanol. The nucleic acid was subsequently allowed to bind to the RNeasy spin column before excess salt was removed by three washing steps using the washing buffers provided. The DENV-2 viral RNA was then eluted with 30 lL of RNase-free water. Finally, DNA was removed using one unit of RQ1 RNase-Free DNase (Promega). The purified RNA was stored at 20 °C. Reverse transcription was first performed in which the cDNA of both the positive and negative viral RNA strands were transcribed with primers (DEN12F: 50 -cttaaatacattcaccaacatggaag-30 and DEN12R: 50 acctgtcatctatgggtttcac-30 ) specifically targeting the DENV-2 viral genome using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Forward primers were used to transcribe cDNA from the negative-strand RNA, whereas reverse primers were used to transcribe cDNA from the positive-strand RNA. Primers and viral RNA were incubated at 70 °C for 5 min and then placed on ice for 2 min. cDNA was then synthesized at 42 °C for 60 min, and then
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Please cite this article in press as: Cheung, Y.Y., et al. Antiviral activity of lanatoside C against dengue virus infection. Antiviral Res. (2014), http:// dx.doi.org/10.1016/j.antiviral.2014.09.007
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heat inactivated at 95 °C for 15 min. The qRT-PCR reaction was performed using the Applied BiosystemsÒ StepOnePlus™ Real-Time PCR system (Life Technologies, Carlsbad, CA, USA) in a mixture containing Maxima™ SYBR Green/ROX qPCR master mix (Roche Applied Science, Indianapolis, IN, USA), forward primer (0.3 lM), reverse primer (0.3 lM), cDNA and nuclease-free water. The
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thermal profile consisted of polymerase activation at 94 °C for 2 min, followed by 40 cycles of PCR (denaturation at 94 °C for 15 s, annealing and extension at 60 °C for 20 s), and melt curve reading. The absolute DENV-2 positive and negative-strand viral RNA copy numbers were then derived from the cycle threshold value by intrapolating from a standard curve.
Fig. 1. Top 20 positive hits from the primary screen for dengue virus infection. HuH-7 cells were infected by DENV-2 at MOI of 1 and treated with the different compounds within the library. Inhibition of DENV-2 was expressed as the percentage of DENV antigen positive cells. Error bars represent the standard deviation.
Fig. 2. Inhibitory effect of lanatoside C on DENV-2 in (A) HuH-7, (B) U937 and (C) HUVEC cells. Cells were infected by DENV-2 at MOI of 1 and treated with the indicated concentrations of lanatoside C. Supernatants were harvested from the infected cells at 48 hpi. Results are mean values from triplicate experiments. Error bars represent the standard error. Statistical analysis was carried out using one-way ANOVA test followed by Dunnett’s post-test (compared against DMSO). ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
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2.7. Viral protein extraction and Western blot
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Cells were lysed with CelLytic™ M Cell Lysis Reagent (Sigma– Aldrich) for protein extraction. The cell lysate was then denatured and proteins were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis at 130 V for 2.5 h. Thereafter, the separated proteins on the gel were transferred onto a nitrocellulose membrane and blocked overnight with 5% w/v non-fat milk. Primary antibodies used were anti-DENV-2 E protein antibody (US Biological), anti-DENV-2 NS5 protein antibody (Genetex) and anti-actin antibody (Millipore) in 1:1000 dilution. Goat anti-rabbit IgG conjugated to horse radish peroxidase (HRP) or goat antimouse IgG-HRP (Thermo Scientific) were used as secondary antibodies in 1:3300 dilution. Chemiluminescence was detected using the SupersignalÒ West Pico Chemiluminescent Substrate (Thermo Scientific). Band intensities were measured using the ImageJ software and expressed as the relative band intensity which is derived from the band intensity of sample/band intensity of b-actin loading control.
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3. Results
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3.1. Drug screening assay
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A drug screen using the US drug library collection, aimed at discovering inhibitory compounds for dengue virus infection was conducted using an immunofluorescence based phenotypic screening assay established previously (Low et al., 2011). The top 20 positive hits displaying greater than 50% inhibition on DENV-2 replication were selected for validation of their anti-DENV activities (Fig. 1). Lanatoside C was found to be a potent inhibitor for DENV-2
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infection and hence was selected for further characterization. Although several compounds showed greater inhibitory activities against DENV-2, they are known to be highly toxic. For instance, both digoxin and digitoxin are known to posses anti-cancer properties in vitro. Studies have shown that the CC50 of both compounds were in the low nM ranges (Prassas et al., 2011). In addition, hycanthone resulted in severe hepatotoxicity and was deemed too toxic for human trials following the death of two patients (Haq et al., 1980).
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3.2. Inhibitory effects of lanatoside C on DENV-2 infection
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Cytotoxicity of lanatoside C was first analyzed in HuH-7, U937 and HUVEC cells to establish suitable concentrations for further studies. Lanatoside C was found to be non-cytotoxic (Fig. 2A–C) to HuH-7, U937 and HUVEC cells at concentrations up to 1.0 lM (HuH-7 and HUVEC cells) and 0.1 lM (U937 cells). Treatment studies using lanatoside C at the established concentration ranges were performed on infected cells to assess the inhibitory effect on DENV-2. As shown in Fig. 2A, lanatoside C displayed dosedependent inhibition on DENV-2 replication in HuH-7 cells at concentrations between 0.1 lM and 0.5 lM (inhibition of 86.84% at 0.5 lM). From the results (Supplementary Fig. 1A and B), lanatoside C inhibited DENV-2 in HuH-7 cells with a 50% inhibitory concentration (IC50) of 0.19 lM, a 50% cytotoxic concentration (CC50) of 5.48 lM and a relatively high selectivity index (SI) (CC50/IC50) of 28.84. In addition, DENV-2 infected U937 cells treated with lanatoside C from 0.01 lM to 0.1 lM also showed dose-dependent inhibition of DENV-2 replication, with a maximum virus inhibition of 79.87% at 0.1 lM (Fig. 2B). The IC50, CC50 and SI of lanatoside C in U937 cells were 0.03 lM, 0.47 lM and
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Fig. 3. Time-of-addition study. HuH-7 cells were infected by DENV-2 at MOI of 1. Lanatoside C was added at the specified time points and the cells were processed for (A) immunofluorescence detection. (B) The percentage of DENV-2 antigen positive cells was also quantified.
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3.5. Decrease in DENV-2 viral protein expression
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Western blot was performed to analyze the effect of lanatoside C on DENV-2 viral protein expression upon infection. Antibodies specific to the DENV-2 structural envelope (E) protein (53 kDa) and non-structural NS5 protein (105 kDa) were used in this part of the study. Both proteins were probed for on the same membrane together with b-actin that was used as a loading control to verify equal loading amongst the samples. Dose-dependent decreases in both DENV-2 viral proteins were observed with increasing concentrations of lanatoside C (Fig. 5A). The relative band intensity ratio of NS5 protein and E protein decreased 6.7 folds and 6.3 folds, respectively, when treated with 1.0 lM lanatoside C (Fig. 5B).
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3.6. Broad spectrum antiviral effects of lanatoside C
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To evaluate the specificity of the antiviral activity of lanatoside C, DENV-1, DENV-3, DENV-4 and another flavivirus – KUNV, alphaviruses – CHIKV and SINV and HEV71 from the Picornaviridae family were evaluated. Lanatoside C was used at a concentration of 0.5 lM or 1.0 lM and these concentrations were shown to be non-cytotoxic to HuH-7, BHK21 and RD cells for the duration of the drug treatment studies (Fig. 6). The inhibitory effects of lanatoside C on the different viruses are shown in Fig. 6. All three dengue serotypes, DENV-1, DENV-3 and DENV-4 virus titres decreased significantly by 86.4%, 95.14% and 73.79% (Fig. 6A),
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To examine the effect of lanatoside C on the synthesis of DENV2 viral RNA, qRT-PCR was performed. The positive and negative DENV-2 viral RNA strands were quantified separately as DENV-2 is a positive-strand RNA virus. It relies on the synthesis of a negative-strand RNA intermediate for replication. Hence, specific quantification of the negative-strand viral RNA can reveal the viral RNA synthesis activity within infected cells (Komurian-Pradel et al., 2004; Bessaud et al., 2008). In addition, any differences in inhibition between the two viral RNA strands could potentially contribute to the understanding of the inhibitory mechanisms of lanatoside C. In this experiment, b-actin was used as an endogenous control to normalize the number of cells in the samples. The standard curve was first generated and subsequently used to determine the absolute DENV-2 viral RNA copy numbers. At both 24 (Fig. 4A) and 48 hpi (Fig. 4B), dose-dependent reduction in the copy numbers of both DENV-2 viral RNA positive-strands and negative-strands were observed with increasing lanatoside C concentrations (Fig. 4A and B).
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In order to narrow down the stage of DENV-2 replication cycle during which lanatoside C exerted its effect, pre- and post-treatment assays were performed. Pre-treatment with lanatoside C showed no inhibition on DENV-2 replication (data not shown). As shown in the images of the immunofluorescence detection, for the post-treatment assay, lanatoside C added at or before 24 hpi abolished DENV-2 replication whereas addition at 48 hpi resulted in loss of inhibition against DENV-2 replication (Fig. 3A). Quantification of the percentage of DENV-2 antigen positive cells (Fig. 3B) corroborated with the results of the immunofluorescence images.
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3.3. Time-of-addition study
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15.67 respectively (Supplementary Fig. 2A and B). Lanatoside C also inhibited DENV-2 replication in HUVEC cells in a dose dependent manner (93.33% at 1.0 lM) with an IC50 of 0.30 lM (Fig. 2C). The CC50 was not calculated for HUVEC as lanatoside C was only tested up to 1.0 lM, which did not affect cell viability.
M oc k
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DENV-2 viral RNA copies (log 10)/mL
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DENV-2 viral RNA copies (log10)/mL
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Post-treatment with lanatoside C (µM) Fig. 4. Quantification of DENV-2 viral RNA strands. Total RNA was extracted at (A) 24 hpi and (B) 48 hpi. Results are mean values from triplicate experiments. Error bars represent the standard deviation. Statistical analysis was carried out using one-way ANOVA test followed by Dunnett’s post-test (compared against DMSO). ⁄ P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
respectively, upon treatment with 1.0 lM of lanatoside C. These results demonstrate that lanatoside C can inhibit the different DENV serotypes. Dose-dependent inhibition of the other viruses tested was observed upon treatment with increasing concentrations of lanatoside C. The virus titre of KUNV (Fig. 6B), HEV71 (Fig. 6C), CHIKV (Fig. 6D) and SINV (Fig. 6E) was reduced by 67.51%, 99.88%, 38.66%, and 72.3%, respectively, at 1.0 lM.
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4. Discussion
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In response to the urgent need for effective anti-DENV drugs, a high-throughput screen was performed to identify anti-DENV compounds. Selected compounds from the primary screen were further examined and amongst them, lanatoside C was recognized to possess anti-DENV-2 activity. Lanatoside C, being relatively novel in the field of antivirals, is a drug commonly used to treat heart diseases. It was recently reported to inhibit various negative-strand RNA viruses (Hoffmann et al., 2008). Being an FDA approved drug also makes lanatoside C an ideal antiviral candidate since it has been approved for human usage. Pre-treatment with lanatoside C did not inhibit DENV-2 replication, ruling out early stages of the DENV-2 replication cycle as possible targets. Time-of-addition study was also performed to narrow down the effective period of lanatoside C. DENV-2 was inhibited only when lanatoside C was added at or before 24 hpi but not at 48 hpi, indicating that the inhibition is completed before 48 hpi. From these results, it was deduced that lanatoside C targets the post-entry stages of the DENV replication cycle such as the
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A Non-structural protein 5 Envelope protein
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Post-treatment with lanatoside C (µM) Fig. 5. Western blot analysis of the DENV-2 structural E protein and non-structural NS5 protein. Total cellular protein was extracted from the cell lysates of DENV-2-infected cells treated with the shown concentrations of lanatoside C. (A) Bands of the specific proteins as observed on the blot. (B) The relative band intensity (calculated from the band intensity of sample/band intensity of b-actin loading control). 0.1% DMSO 0.5 M lanatoside C 1.0 M lanatoside C
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Fig. 6. Inhibitory effect of lanatoside C on other positive-sense RNA viruses. (A) HuH-7 cells were infected with DENV-1, -3 and -4 at MOI of 1. (B) HuH-7 cells were infected with KUNV at MOI of 1. (C) RD cells were infected with HEV71 at MOI of 1. (D) BHK21 cells were infected with CHIKV at MOI of 1. (E) BHK21 cells were infected with SINV at MOI of 1. Infected cells were then treated with 0.5 lM and 1.0 lM of lanatoside C. Supernatants were harvested from the infected cells at the respective time post infection. Virus titre is compared against the 0.1% DMSO control for each virus. Results are the mean values from triplicate experiments with error bars representing the standard error. Statistical analysis was carried out using one-way ANOVA test followed by Dunnett’s post-test (compared against DMSO). ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
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translation of viral proteins, viral replication complex assembly, and viral RNA synthesis. Lanatoside C was also shown to effectively inhibit both DENV-2 viral RNA synthesis and viral protein expression. This may suggest that the drug acts on stages before
the assembly of progeny virus, hence ruling out the late stages of virus replication as probable targets. Therefore, a possible target step is the viral RNA synthesis. Interference during viral RNA synthesis is coupled to the observed decline in both negative and
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positive DENV-2 viral RNA strands from which viral proteins are massively translated. This would then lead to the reduction in DENV-2 viral proteins and the drop in DENV-2 viral titre. In this study, HuH-7 cells were the main cell line used. U937 cell line substitutes the high maintenance peripheral blood mononuclear cells (PBMC) which are good representatives of in vivo infection (Clyde et al., 2006). HUVEC cells, a human umbilical vein endothelial cell line, were also tested as they are a representative of non-cancerous, primary cell type. Inhibition of DENV-2 replication in HUVEC demonstrates that the inhibitory effect of lanatoside C is not caused by its anti-cancer effects. DENV-2 was inhibited dose-dependently with increasing lanatoside C in all three cell lines although inhibition occurred in U937 cells at lower drug concentrations. However, the drop in cell viability of U937 cells restricted its use at higher lanatoside C concentrations. Nevertheless, these demonstrate that lanatoside C effectively inhibits DENV-2 in the various cell types targeted by the virus. Potent inhibition against the all four DENV serotypes, KUNV, CHIKV, SINV and HEV71 indicates that lanatoside C is able to inhibit positive-sense RNA viruses of different families. Differences in the viral replication cycles and compensatory mechanisms of the distinct viruses may account for the variations in susceptibility of different viruses to lanatoside C. As observed in our study and that of Hoffmann et al. (2008), the antiviral activity of lanatoside C was relatively broad-spectrum. This suggests that the drug targets cellular factors rather than viral proteins and this is advantageous since emergence of resistance is less likely to occur. Inhibition of the cellular Na+-K+-ATPase pump may contribute to the antiviral properties of lanatoside C. Intracellular Na+ and K+ levels are important for the transport of solutes across cellular membranes and may alter cellular activities that can affect virus replication or activate host antiviral mechanisms. It was reported that viral replication was indeed affected under conditions of raised intracellular Na+ and reduced intracellular K+ levels (Nagai et al., 1972; Hartley et al., 1993; Chen et al., 2004; Hoffmann et al., 2008; Bertol et al., 2011). To this end, much more work would be needed to elucidate the exact mechanism of inhibition by lanatoside C and its association with the DENV virus replication cycle. Further downstream studies in suitable murine models would also be required to evaluate the reproducibility of the inhibitory effects of lanatoside C in vivo. In conclusion, our study has successfully demonstrated the inhibition of DENV-2 replication by lanatoside C with minimal cytotoxicity. Lanatoside C might potentially be developed as broad-spectrum antiviral targeting viruses probably sharing the common steps targeted by the compound. Hence, these warrant lanatoside C as a promising antiviral drug that should be further examined.
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Conflict of interest
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Acknowledgements
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Q3 This work was supported by NUS Start-up grant (R182-000- Q4 165-133) and MINDEF DIRP grant (R182-000210-232).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral.2014.09. 007.
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References
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Bertol, J.W., Rigotto, C., de Pádua, R.M., Kreis, W., Barardi, C.R.M., Braga, F.C., Simões, C.M.O., 2011. Antiherpes activity of glucoevatromonoside, a cardenolide isolated from a Brazilian cultivar of Digitalis lanata. Antiviral Res. 92, 73–80. Bessaud, M., Autret, A., Jegouic, S., Balanant, J., Joffret, M.L., Delpeyroux, F., 2008. Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: evidence for false-priming and improvement by tagged RT-PCR. J. Virol. Methods 153, 182–189. Chen, X.-J., Seth, S., Yue, G., Kamat, P., Compans, R.W., Guidot, D., Brown, L.A., Eaton, D.C., Jain, L., 2004. Influenza virus inhibits ENaC and lung fluid clearance. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L366–L373. Clyde, K., Kyle, J.L., Harris, E., 2006. Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J. Virol. 80, 11418– 11431. Diamond, M.S., Zachariah, M., Harris, E., 2002. Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA. Virology 304, 211–221. Dodson, A.W., Taylor, T.J., Knipe, D.M., Coen, D.M., 2007. Inhibitors of the sodium potassium ATPase that impair herpes simplex virus replication identified via a chemical screening approach. Virology 366, 340–348. Haq, M.M., Legha, S.S., Wiseman, C.W., Bodey, G.P., 1980. Hepatotoxicity of hycanthone in patients with metastatic breast cancer. Cancer Treat. Rep. 64, 929–932. Hartley, C.E., Buchan, A., Randall, S., Skinner, G.R.B., Osborne, M., Tomkins, L.M., 1993. The effects of lithium and potassium on macromolecular synthesis in herpes simplex virus-infected cells. J. Gen. Virol. 74, 1519–1525. Hoffmann, H.H., Palese, P., Shaw, M.L., 2008. Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity. Antiviral Res. 80, 124–134. Komurian-Pradel, F., Perret, M., Deiman, B., Sodoyer, M., Lotteau, V., ParanhosBaccala, G., Andre, P., 2004. Strand specific quantitative real-time PCR to study replication of hepatitis C virus genome. J. Virol. Methods 116, 103–106. Lim, S.P., Wang, Q.-Y., Noble, C.G., Chen, Y.-L., Dong, H., Zou, B., Yokokawa, F., Nilar, S., Smith, P., Beer, D., Lescar, J., Shi, P.-Y., 2013. Ten years of dengue drug discovery: progress and prospects. Antiviral Res. 100, 500–519. Low, J.S., Wu, K.X., Chen, K.C., Ng, M.M., Chu, J.J., 2011. Narasin, a novel antiviral compound that blocks dengue virus protein expression. Antivir. Ther. 16, 1203– 1218. Mukhopadhyay, S., Kuhn, R.J., Rossmann, M.G., 2005. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3, 13–22. Nagai, Y., Maeno, K., Iinuma, M., Yoshida, T., Matsumoto, T., 1972. Inhibition of virus growth by ouabain: effect of ouabain on the growth of HVJ in chick embryo cells. J. Virol. 9, 234–243. Prassas, I., Karagiannis, G.S., Batruch, I., Dimitromanolakis, A., Datti, A., Diamandis, E.P., 2011. Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks. Mol. Cancer Ther. 10, 2083– 2093. Takhampunya, R., Ubol, S., Houng, H.-S., Cameron, C.E., Padmanabhan, R., 2006. Inhibition of dengue virus replication by mycophenolic acid and ribavirin. J. Gen. Virol. 87, 1947–1952. Tomita, Y., Kuwata, T., 1978. Suppression of murine leukaemia virus production by ouabain and interferon in mouse cells. J. Gen. Virol. 38, 223–230.
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None declared.
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Antiviral activity of lanatoside C against dengue virus infection
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Yan Yi Cheung a, Karen Caiyun Chen a, Huixin Chen a, Eng Khuan Seng b, Justin Jang Hann Chu a,⇑ a Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore b School of Chemical & Life Sciences, 180 Ang Mo Kio Ave 8, Nanyang Polytechnic, Singapore 569830, Singapore
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Article history: Received 18 March 2014 Revised 7 August 2014 Accepted 13 September 2014 Available online xxxx Keywords: Dengue virus Cardiac glycoside Flavivirus Lanatoside C Antiviral compounds
a b s t r a c t Dengue infection poses a serious threat globally due to its recent rapid spread and rise in incidence. Currently, there is no approved vaccine or effective antiviral drug for dengue virus infection. In response to the urgent need for the development of an effective antiviral for dengue virus, the US Drug Collection library was screened in this study to identify compounds with anti-dengue activities. Lanatoside C, an FDA approved cardiac glycoside was identified as a candidate anti-dengue compound. Our data revealed that lanatoside C has an IC50 of 0.19 lM for dengue virus infection in HuH-7 cells. Dose-dependent reduction in dengue viral RNA and viral proteins synthesis were also observed upon treatment with increasing concentrations of lanatoside C. Time of addition study indicated that lanatoside C inhibits the early processes of the dengue virus replication cycle. Furthermore, lanatoside C can effectively inhibit all four serotypes of dengue virus, flavivirus Kunjin, alphavirus Chikungunya and Sindbis virus as well as the human enterovirus 71. These findings suggest that lanatoside C possesses broad spectrum antiviral activity against several groups of positive-sense RNA viruses. Ó 2014 Published by Elsevier B.V.
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1. Introduction
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Dengue virus (DENV), the most significant virus in the Flaviviridae family with the highest morbidity and mortality rates, poses a serious global threat in the past few decades. DENV is serologically subdivided into four serotypes, DENV-1, -2, -3 and -4. The enveloped virus contains a single-stranded, positive-sense RNA genome that is directly translated into a single polyprotein which is eventually cleaved into the ten individual viral proteins. The three structural proteins, envelope (E), capsid (C) and premembrane (prM) proteins form the virion particles (Mukhopadhyay et al., 2005). The remaining seven non-structural proteins are primarily involved in viral RNA replication within the infected cells. Dengue infection can manifest itself either as a mild, self-limiting clinical infection or the more severe dengue hemorrhagic fever (DHF) and even the life-threatening dengue shock syndrome (DSS). Currently, there are numerous candidate compounds (Lim et al., 2013), including the well-known mycophenolic acid and ribavirin (Diamond et al., 2002; Takhampunya et al., 2006) that are reported
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⇑ Corresponding author at: Laboratory of Molecular RNA Virology and Antiviral Strategies, Yong Loo Lin School of Medicine, Department of Microbiology, MD4, Level 5, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore. Tel.: +65 65163278; fax: +65 67766872. E-mail address: [email protected] (J.J.H. Chu).
to possess anti-DENV activities. Nevertheless, none of them are approved yet for the treatment of the disease. Therefore, the development of safe and effective antiviral treatments is now a pressing issue to combat this medically important mosquito-borne viral pathogen. Lanatoside C, a US Food and Drug Administration (FDA) approved cardiac glycoside that acts by inhibiting the Na+-K+-ATPase pump was recently found to inhibit several negative-strand RNA viruses including the influenza virus, vesicular stomatitis virus and Newcastle disease virus (Hoffmann et al., 2008). Another well studied cardiac glycoside, ouabain that acts similarly by inhibiting the Na+-K+-ATPase pump also inhibited herpes simplex virus (Dodson et al., 2007), Sendai virus (Nagai et al., 1972; Tomita and Kuwata, 1978) and murine leukemia virus (Tomita and Kuwata, 1978). Increased intracellular Na+ and reduced intracellular K+ concentrations have both been demonstrated to affect replication of several negative-strand viruses, DNA viruses and retroviruses (Nagai et al., 1972; Hartley et al., 1993; Chen et al., 2004; Hoffmann et al., 2008; Bertol et al., 2011). In our high-throughput screen of the US Drug Collection library for anti-DENV compounds, lanatoside C was identified as a potent inhibitor of dengue virus infection. The inhibitory effect of lanatoside C on DENV-2 was examined by measuring the production of infectious virus particles and viral RNA as well as viral protein expression. The study was also extended to the different DENV
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serotypes and other positive-strand RNA viruses (Kunjin virus, Chikungunya virus, Sindbis virus and Human enterovirus 71). Our data showed dose-dependent inhibition of DENV-2 virus infection, viral RNA production and viral protein synthesis following treatment with increasing concentrations of lanatoside C. Other viruses within the Flaviviridae, Togaviridae and Picornaviridae families were also found to be inhibited by lanatoside C.
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2. Materials and methods
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2.1. Cell lines and viruses
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Human umbilical vein endothelial (HUVEC) cells were purchased from Clonetics, USA. Baby hamster kidney fibroblast (BHK21) cells, human muscle rhabdomyosarcoma (RD) cells and mosquito cell line C6/36 from Aedes albopictus were obtained from American Type Culture Collection (ATCC). The human leukemic monocyte lymphoma (U937) cell line was kindly provided by Professor Ng Mah Lee, Department of Microbiology, National University of Singapore (NUS). The human hepatoma (HuH-7) cell line was a kind gift from Dr. Priscilla Yang, Department of Microbiology and Immunology, Harvard Medical School. HUVEC, BHK21 and U937 cells were maintained in RPMI-1640 medium (Sigma– Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS). HuH-7 and RD cells were maintained in DMEM (Sigma–Aldrich) supplemented with 10% FCS. C6/36 cells were maintained in L-15 medium (Sigma–Aldrich) supplemented with 10% heat inactivated FCS. BHK21, U937, RD and HuH-7 cells were maintained at 37 °C with 5% CO2 in a humidified incubator. C6/36 cells were maintained at 28 °C, in the absence of CO2. DENV serotype 1–4 (Singapore isolates) were grown in C6/36 mosquito cell line derived from A. albopictus. Sindbis virus (SINV) and Kunjin virus (KUNV) were kind gifts from Professor Ng Mah Lee, Department of Microbiology, National University of Singapore. Human Enterovirus 71 (5865/SIN/000009) (HEV71) was kindly provided by A/P Vincent Chow, Department of Microbiology, National University of Singapore and Chikungunya virus (D1225Y08) (CHIKV) was kindly provided Dr. Ng Lee Ching, Environmental Health Institute, National Environment Agency.
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2.2. US drug collection screen
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The US Drug Collection (Microsource, Discovery Systems Inc., Gaylordsville, CT, USA) library consisting of a total of 1040 compounds was screened for compounds that effectively inhibit DENV infection. Briefly, HuH-7 cells were infected with DENV-2 at multiplicity of infection (MOI) of 1 and incubated at 37 °C for 1 h to allow virus adsorption. The cells were then incubated with the different compounds and subsequently processed for immunofluorescence assay as described previously (Low et al., 2011). Inhibition of DENV-2 by the various compounds, relative to the DMSO-treated control was expressed as the percentage of DENV antigen positive cells. The top 20 positive hits displaying greater than 50% inhibition against DENV-2 were selected for further validation of their anti-DENV properties. HuH-7 cells were infected and treated with the selected compounds for validation. The supernatant was collected for quantification of infectious viral titre via plaque assay.
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2.3. Cell viability assay
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AlamarBlueÒ cell viability assay (Invitrogen, Carlsbad, CA, USA) was used to measure the cell viability of the various cell lines following treatment with different concentrations of lanatoside C (Sigma–Aldrich) following manufacturer’s instructions. Sodium azide and 0.1% DMSO were used as experimental controls. The
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fluorescence intensity was measured by the Infinite M200 microplate reader (Tecan, Männerdorf, Switzerland) using excitation and emission wavelengths of 570 nm and 585 nm, respectively. The data was then processed by the i-control 1.6 software (Tecan).
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2.4. Dose-dependent studies
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2.4.1. Infection of cells For DENV-1, DENV-2, DENV-3, DENV-4 and Kunjin virus (KUNV), adherent HuH-7 and HUVEC cells were seeded on 24-well plates at 5 104 and 1 105 cells/well one day prior to infection. Cells were then infected with the different viruses at MOI of 1. For the suspension cell line, U937, cells were seeded at 8 105 cells/well on the day of infection before being infected by DENV-2 (MOI of 1). The cells were then incubated at 37 °C for 1 h to allow virus adsorption. For CHIKV and SINV virus, BHK21 cells were seeded on 24-well plates at 7.5 104 cells/well one day prior to infection. The cells were infected at MOI of 1 as described above for 1 h (SINV) and 1.5 h (CHIKV). For HEV71, RD cells were seeded on 24-well plates at 2 105 cells/well one day prior to infection. The cells were infected at MOI of 1 as described above for 1 h. Following infection, the cells were rinsed with PBS before treatment with lanatoside C.
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2.4.2. Lanatoside C treatment Appropriate volumes of medium (supplemented with 2% FCS) containing various concentrations of drug were added to the infected cells. 0.1% DMSO was used as the vehicle control for comparison with the drug-treated infected cells. The cells were then incubated at 37 °C for either 12 h (SINV and HEV71 infected BHK21 and RD cells), 24 h (CHIKV infected BHK21 cells), 48 h (DENV-1, DENV-2, DENV-3, DENV-4 and KUNV infected HuH-7, HUVEC and U937 cells) before they were harvested for plaque assay.
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2.5. Time-of-addition study
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For the pretreatment assay, HuH-7 cells were treated with lanatoside C for 2 h before infection by DENV-2. For the time-ofaddition study, HuH-7 cells were first infected with DENV-2 at MOI of 1 and lanatoside C was then added at 0, 4, 6, 8, 10, 12, 24 and 48 hpi. The infected cells were processed for immunofluorescence detection at 72 hpi and the percentage of DENV antigen positive cells were quantified.
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2.6. Quantitative RT-PCR
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At 24 and 48 hpi, DENV-2 viral RNA was extracted from the samples, using the RNeasy Mini Kit (QIAGEN), following the manufacturer’s protocols. Briefly, the nucleic acid was first precipitated using 100% ethanol. The nucleic acid was subsequently allowed to bind to the RNeasy spin column before excess salt was removed by three washing steps using the washing buffers provided. The DENV-2 viral RNA was then eluted with 30 lL of RNase-free water. Finally, DNA was removed using one unit of RQ1 RNase-Free DNase (Promega). The purified RNA was stored at 20 °C. Reverse transcription was first performed in which the cDNA of both the positive and negative viral RNA strands were transcribed with primers (DEN12F: 50 -cttaaatacattcaccaacatggaag-30 and DEN12R: 50 acctgtcatctatgggtttcac-30 ) specifically targeting the DENV-2 viral genome using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Forward primers were used to transcribe cDNA from the negative-strand RNA, whereas reverse primers were used to transcribe cDNA from the positive-strand RNA. Primers and viral RNA were incubated at 70 °C for 5 min and then placed on ice for 2 min. cDNA was then synthesized at 42 °C for 60 min, and then
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heat inactivated at 95 °C for 15 min. The qRT-PCR reaction was performed using the Applied BiosystemsÒ StepOnePlus™ Real-Time PCR system (Life Technologies, Carlsbad, CA, USA) in a mixture containing Maxima™ SYBR Green/ROX qPCR master mix (Roche Applied Science, Indianapolis, IN, USA), forward primer (0.3 lM), reverse primer (0.3 lM), cDNA and nuclease-free water. The
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thermal profile consisted of polymerase activation at 94 °C for 2 min, followed by 40 cycles of PCR (denaturation at 94 °C for 15 s, annealing and extension at 60 °C for 20 s), and melt curve reading. The absolute DENV-2 positive and negative-strand viral RNA copy numbers were then derived from the cycle threshold value by intrapolating from a standard curve.
Fig. 1. Top 20 positive hits from the primary screen for dengue virus infection. HuH-7 cells were infected by DENV-2 at MOI of 1 and treated with the different compounds within the library. Inhibition of DENV-2 was expressed as the percentage of DENV antigen positive cells. Error bars represent the standard deviation.
Fig. 2. Inhibitory effect of lanatoside C on DENV-2 in (A) HuH-7, (B) U937 and (C) HUVEC cells. Cells were infected by DENV-2 at MOI of 1 and treated with the indicated concentrations of lanatoside C. Supernatants were harvested from the infected cells at 48 hpi. Results are mean values from triplicate experiments. Error bars represent the standard error. Statistical analysis was carried out using one-way ANOVA test followed by Dunnett’s post-test (compared against DMSO). ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
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2.7. Viral protein extraction and Western blot
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Cells were lysed with CelLytic™ M Cell Lysis Reagent (Sigma– Aldrich) for protein extraction. The cell lysate was then denatured and proteins were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis at 130 V for 2.5 h. Thereafter, the separated proteins on the gel were transferred onto a nitrocellulose membrane and blocked overnight with 5% w/v non-fat milk. Primary antibodies used were anti-DENV-2 E protein antibody (US Biological), anti-DENV-2 NS5 protein antibody (Genetex) and anti-actin antibody (Millipore) in 1:1000 dilution. Goat anti-rabbit IgG conjugated to horse radish peroxidase (HRP) or goat antimouse IgG-HRP (Thermo Scientific) were used as secondary antibodies in 1:3300 dilution. Chemiluminescence was detected using the SupersignalÒ West Pico Chemiluminescent Substrate (Thermo Scientific). Band intensities were measured using the ImageJ software and expressed as the relative band intensity which is derived from the band intensity of sample/band intensity of b-actin loading control.
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3. Results
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3.1. Drug screening assay
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A drug screen using the US drug library collection, aimed at discovering inhibitory compounds for dengue virus infection was conducted using an immunofluorescence based phenotypic screening assay established previously (Low et al., 2011). The top 20 positive hits displaying greater than 50% inhibition on DENV-2 replication were selected for validation of their anti-DENV activities (Fig. 1). Lanatoside C was found to be a potent inhibitor for DENV-2
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infection and hence was selected for further characterization. Although several compounds showed greater inhibitory activities against DENV-2, they are known to be highly toxic. For instance, both digoxin and digitoxin are known to posses anti-cancer properties in vitro. Studies have shown that the CC50 of both compounds were in the low nM ranges (Prassas et al., 2011). In addition, hycanthone resulted in severe hepatotoxicity and was deemed too toxic for human trials following the death of two patients (Haq et al., 1980).
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3.2. Inhibitory effects of lanatoside C on DENV-2 infection
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Cytotoxicity of lanatoside C was first analyzed in HuH-7, U937 and HUVEC cells to establish suitable concentrations for further studies. Lanatoside C was found to be non-cytotoxic (Fig. 2A–C) to HuH-7, U937 and HUVEC cells at concentrations up to 1.0 lM (HuH-7 and HUVEC cells) and 0.1 lM (U937 cells). Treatment studies using lanatoside C at the established concentration ranges were performed on infected cells to assess the inhibitory effect on DENV-2. As shown in Fig. 2A, lanatoside C displayed dosedependent inhibition on DENV-2 replication in HuH-7 cells at concentrations between 0.1 lM and 0.5 lM (inhibition of 86.84% at 0.5 lM). From the results (Supplementary Fig. 1A and B), lanatoside C inhibited DENV-2 in HuH-7 cells with a 50% inhibitory concentration (IC50) of 0.19 lM, a 50% cytotoxic concentration (CC50) of 5.48 lM and a relatively high selectivity index (SI) (CC50/IC50) of 28.84. In addition, DENV-2 infected U937 cells treated with lanatoside C from 0.01 lM to 0.1 lM also showed dose-dependent inhibition of DENV-2 replication, with a maximum virus inhibition of 79.87% at 0.1 lM (Fig. 2B). The IC50, CC50 and SI of lanatoside C in U937 cells were 0.03 lM, 0.47 lM and
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Fig. 3. Time-of-addition study. HuH-7 cells were infected by DENV-2 at MOI of 1. Lanatoside C was added at the specified time points and the cells were processed for (A) immunofluorescence detection. (B) The percentage of DENV-2 antigen positive cells was also quantified.
Please cite this article in press as: Cheung, Y.Y., et al. Antiviral activity of lanatoside C against dengue virus infection. Antiviral Res. (2014), http:// dx.doi.org/10.1016/j.antiviral.2014.09.007
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3.5. Decrease in DENV-2 viral protein expression
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Western blot was performed to analyze the effect of lanatoside C on DENV-2 viral protein expression upon infection. Antibodies specific to the DENV-2 structural envelope (E) protein (53 kDa) and non-structural NS5 protein (105 kDa) were used in this part of the study. Both proteins were probed for on the same membrane together with b-actin that was used as a loading control to verify equal loading amongst the samples. Dose-dependent decreases in both DENV-2 viral proteins were observed with increasing concentrations of lanatoside C (Fig. 5A). The relative band intensity ratio of NS5 protein and E protein decreased 6.7 folds and 6.3 folds, respectively, when treated with 1.0 lM lanatoside C (Fig. 5B).
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3.6. Broad spectrum antiviral effects of lanatoside C
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To evaluate the specificity of the antiviral activity of lanatoside C, DENV-1, DENV-3, DENV-4 and another flavivirus – KUNV, alphaviruses – CHIKV and SINV and HEV71 from the Picornaviridae family were evaluated. Lanatoside C was used at a concentration of 0.5 lM or 1.0 lM and these concentrations were shown to be non-cytotoxic to HuH-7, BHK21 and RD cells for the duration of the drug treatment studies (Fig. 6). The inhibitory effects of lanatoside C on the different viruses are shown in Fig. 6. All three dengue serotypes, DENV-1, DENV-3 and DENV-4 virus titres decreased significantly by 86.4%, 95.14% and 73.79% (Fig. 6A),
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To examine the effect of lanatoside C on the synthesis of DENV2 viral RNA, qRT-PCR was performed. The positive and negative DENV-2 viral RNA strands were quantified separately as DENV-2 is a positive-strand RNA virus. It relies on the synthesis of a negative-strand RNA intermediate for replication. Hence, specific quantification of the negative-strand viral RNA can reveal the viral RNA synthesis activity within infected cells (Komurian-Pradel et al., 2004; Bessaud et al., 2008). In addition, any differences in inhibition between the two viral RNA strands could potentially contribute to the understanding of the inhibitory mechanisms of lanatoside C. In this experiment, b-actin was used as an endogenous control to normalize the number of cells in the samples. The standard curve was first generated and subsequently used to determine the absolute DENV-2 viral RNA copy numbers. At both 24 (Fig. 4A) and 48 hpi (Fig. 4B), dose-dependent reduction in the copy numbers of both DENV-2 viral RNA positive-strands and negative-strands were observed with increasing lanatoside C concentrations (Fig. 4A and B).
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DENV-2 (+) RNA
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Post-treatment with lanatoside C (µM)
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In order to narrow down the stage of DENV-2 replication cycle during which lanatoside C exerted its effect, pre- and post-treatment assays were performed. Pre-treatment with lanatoside C showed no inhibition on DENV-2 replication (data not shown). As shown in the images of the immunofluorescence detection, for the post-treatment assay, lanatoside C added at or before 24 hpi abolished DENV-2 replication whereas addition at 48 hpi resulted in loss of inhibition against DENV-2 replication (Fig. 3A). Quantification of the percentage of DENV-2 antigen positive cells (Fig. 3B) corroborated with the results of the immunofluorescence images.
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DENV-2 (-) RNA
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15.67 respectively (Supplementary Fig. 2A and B). Lanatoside C also inhibited DENV-2 replication in HUVEC cells in a dose dependent manner (93.33% at 1.0 lM) with an IC50 of 0.30 lM (Fig. 2C). The CC50 was not calculated for HUVEC as lanatoside C was only tested up to 1.0 lM, which did not affect cell viability.
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DENV-2 viral RNA copies (log 10)/mL
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DENV-2 viral RNA copies (log10)/mL
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Post-treatment with lanatoside C (µM) Fig. 4. Quantification of DENV-2 viral RNA strands. Total RNA was extracted at (A) 24 hpi and (B) 48 hpi. Results are mean values from triplicate experiments. Error bars represent the standard deviation. Statistical analysis was carried out using one-way ANOVA test followed by Dunnett’s post-test (compared against DMSO). ⁄ P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
respectively, upon treatment with 1.0 lM of lanatoside C. These results demonstrate that lanatoside C can inhibit the different DENV serotypes. Dose-dependent inhibition of the other viruses tested was observed upon treatment with increasing concentrations of lanatoside C. The virus titre of KUNV (Fig. 6B), HEV71 (Fig. 6C), CHIKV (Fig. 6D) and SINV (Fig. 6E) was reduced by 67.51%, 99.88%, 38.66%, and 72.3%, respectively, at 1.0 lM.
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4. Discussion
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In response to the urgent need for effective anti-DENV drugs, a high-throughput screen was performed to identify anti-DENV compounds. Selected compounds from the primary screen were further examined and amongst them, lanatoside C was recognized to possess anti-DENV-2 activity. Lanatoside C, being relatively novel in the field of antivirals, is a drug commonly used to treat heart diseases. It was recently reported to inhibit various negative-strand RNA viruses (Hoffmann et al., 2008). Being an FDA approved drug also makes lanatoside C an ideal antiviral candidate since it has been approved for human usage. Pre-treatment with lanatoside C did not inhibit DENV-2 replication, ruling out early stages of the DENV-2 replication cycle as possible targets. Time-of-addition study was also performed to narrow down the effective period of lanatoside C. DENV-2 was inhibited only when lanatoside C was added at or before 24 hpi but not at 48 hpi, indicating that the inhibition is completed before 48 hpi. From these results, it was deduced that lanatoside C targets the post-entry stages of the DENV replication cycle such as the
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Post-treatment with lanatoside C (µM) Fig. 5. Western blot analysis of the DENV-2 structural E protein and non-structural NS5 protein. Total cellular protein was extracted from the cell lysates of DENV-2-infected cells treated with the shown concentrations of lanatoside C. (A) Bands of the specific proteins as observed on the blot. (B) The relative band intensity (calculated from the band intensity of sample/band intensity of b-actin loading control). 0.1% DMSO 0.5 M lanatoside C 1.0 M lanatoside C
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Fig. 6. Inhibitory effect of lanatoside C on other positive-sense RNA viruses. (A) HuH-7 cells were infected with DENV-1, -3 and -4 at MOI of 1. (B) HuH-7 cells were infected with KUNV at MOI of 1. (C) RD cells were infected with HEV71 at MOI of 1. (D) BHK21 cells were infected with CHIKV at MOI of 1. (E) BHK21 cells were infected with SINV at MOI of 1. Infected cells were then treated with 0.5 lM and 1.0 lM of lanatoside C. Supernatants were harvested from the infected cells at the respective time post infection. Virus titre is compared against the 0.1% DMSO control for each virus. Results are the mean values from triplicate experiments with error bars representing the standard error. Statistical analysis was carried out using one-way ANOVA test followed by Dunnett’s post-test (compared against DMSO). ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
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translation of viral proteins, viral replication complex assembly, and viral RNA synthesis. Lanatoside C was also shown to effectively inhibit both DENV-2 viral RNA synthesis and viral protein expression. This may suggest that the drug acts on stages before
the assembly of progeny virus, hence ruling out the late stages of virus replication as probable targets. Therefore, a possible target step is the viral RNA synthesis. Interference during viral RNA synthesis is coupled to the observed decline in both negative and
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positive DENV-2 viral RNA strands from which viral proteins are massively translated. This would then lead to the reduction in DENV-2 viral proteins and the drop in DENV-2 viral titre. In this study, HuH-7 cells were the main cell line used. U937 cell line substitutes the high maintenance peripheral blood mononuclear cells (PBMC) which are good representatives of in vivo infection (Clyde et al., 2006). HUVEC cells, a human umbilical vein endothelial cell line, were also tested as they are a representative of non-cancerous, primary cell type. Inhibition of DENV-2 replication in HUVEC demonstrates that the inhibitory effect of lanatoside C is not caused by its anti-cancer effects. DENV-2 was inhibited dose-dependently with increasing lanatoside C in all three cell lines although inhibition occurred in U937 cells at lower drug concentrations. However, the drop in cell viability of U937 cells restricted its use at higher lanatoside C concentrations. Nevertheless, these demonstrate that lanatoside C effectively inhibits DENV-2 in the various cell types targeted by the virus. Potent inhibition against the all four DENV serotypes, KUNV, CHIKV, SINV and HEV71 indicates that lanatoside C is able to inhibit positive-sense RNA viruses of different families. Differences in the viral replication cycles and compensatory mechanisms of the distinct viruses may account for the variations in susceptibility of different viruses to lanatoside C. As observed in our study and that of Hoffmann et al. (2008), the antiviral activity of lanatoside C was relatively broad-spectrum. This suggests that the drug targets cellular factors rather than viral proteins and this is advantageous since emergence of resistance is less likely to occur. Inhibition of the cellular Na+-K+-ATPase pump may contribute to the antiviral properties of lanatoside C. Intracellular Na+ and K+ levels are important for the transport of solutes across cellular membranes and may alter cellular activities that can affect virus replication or activate host antiviral mechanisms. It was reported that viral replication was indeed affected under conditions of raised intracellular Na+ and reduced intracellular K+ levels (Nagai et al., 1972; Hartley et al., 1993; Chen et al., 2004; Hoffmann et al., 2008; Bertol et al., 2011). To this end, much more work would be needed to elucidate the exact mechanism of inhibition by lanatoside C and its association with the DENV virus replication cycle. Further downstream studies in suitable murine models would also be required to evaluate the reproducibility of the inhibitory effects of lanatoside C in vivo. In conclusion, our study has successfully demonstrated the inhibition of DENV-2 replication by lanatoside C with minimal cytotoxicity. Lanatoside C might potentially be developed as broad-spectrum antiviral targeting viruses probably sharing the common steps targeted by the compound. Hence, these warrant lanatoside C as a promising antiviral drug that should be further examined.
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Conflict of interest
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Acknowledgements
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Q3 This work was supported by NUS Start-up grant (R182-000- Q4 165-133) and MINDEF DIRP grant (R182-000210-232).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral.2014.09. 007.
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None declared.
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