The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae)

Donald, Claire L.

Kohl, Alain

Merits, Andres

Royle, Jamie

Varjak, Margus

English

Jamie Royle

Claire L. Donald

Andres Merits

Alain Kohl

Margus Varjak

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The Veterinary Journal

Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses

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The Veterinary Journal 230 (2017) 62–64Differential effects of lipid biosynthesis inhibitors on Zika and SemlikiForest virusesJamie Roylea, Claire L. Donalda, Andres Meritsb, Alain Kohla, Margus Varjaka,*aMRC -University of Glasgow Centre for Virus Research, Glasgow G61 1QH, Scotland,UKb Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaA R T I C L E I N F OKeywords:Zika virusSemliki Forest virusLipid biosynthesisAntiviral agentA B S T R A C TThe recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to thispreviously neglected mosquito-borne pathogen and the need for efﬁcient therapies. Since ﬂavivirusreplication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway,5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virusreplication. At concentrations previously shown to inhibit the replication of other ﬂaviviruses, neitherdrug had a signiﬁcant antiviral affect against ZIKV, but reduced the replication of the non-relatedmosquito-borne Semliki Forest virus (Togaviridae).© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).Contents lists available at ScienceDirectThe Veterinary Journaljourna l homepage: www.e lsev ier .com/ locate / tv j lArboviruses are a diverse group of viruses that are transmittedby arthropod vectors, including mosquitoes, ticks and midges andbelong to four main viral families (Togaviridae, Flaviviridae,Bunyaviridae and Reoviridae). In the Flaviviridae (genus Flavivirus),important and/or representative viruses include Zika virus (ZIKV)and dengue virus (DENV), whilst the genus Alphavirus in theTogaviridae family contains chikungunya virus (CHIKV) and SemlikiForest virus (SFV). Arbovirus infections can cause severe disease ina range of human and animal hosts, resulting in a signiﬁcant socialand economic impact in many areas of the world (Weaver andReisen, 2010).The recent outbreak of infection with Zika virus (ZIKV,Flaviviridae) in human beings has brought increased attention topreviously this neglected arboviruses. ZIKV is thought to be mainlyspread by Aedes aegypti mosquitoes in the South Americanoutbreak (Ferreira-de-Brito et al., 2016). Symptoms of infectionin human beings are generally mild, consisting of fever and jointpain. However, infection during pregnancy can cause microcephalyand other manifestations that are grouped as Zika congenitalsyndrome, while infection in adults has been linked to Guillain-Barré syndrome (Russo et al., 2017).The replication of several ﬂaviviruses, including DENV andalphaviruses (CHIKV), is dependent on continuous synthesis offatty acids; the use of chemicals to inhibit their production hasbeen shown to decrease virus production (Heaton et al., 2010;* Corresponding author.E-mail address: margus.varjak@glasgow.ac.uk (M. Varjak).https://doi.org/10.1016/j.tvjl.2017.10.0091090-0233/© 2017 The Authors. Published by Elsevier Ltd. This is an open access articKarlas et al., 2016). These pathways are an attractive target forantiviral agents, as has been postulated for ﬂaviviruses, includingZIKV (Martin-Acebes et al., 2016). Fatty acid synthesis in thecytoplasm of eukaryotic cells requires three different enzymes thatact in an orchestrated manner: ATP citrate lyase (ACLY), acetyl-CoAcarboxylase (ACC) and fatty acid synthase (FASN). Here we aimedto assess if ZIKV replication could be restricted through thetreatment of infected cells with drugs that inhibit fatty acidbiosynthesis. To this end we used 5-(tetradecyloxyl)-2-furoic acid(TOFA; Sigma–Aldrich) and cerulenin (Sigma–Aldrich), whichinhibit ACC and FASN, respectively.Firstly, the toxicity of the drugs was tested on adenocarcinomichuman alveolar basal epithelial (A549) cells grown in Dulbecco’smodiﬁed Eagle’s medium (DMEM) supplemented with 10% foetalbovine serum. Cell viability was assessed by measuring ATP levelsusing the CellTitre-Glo Luminescent Cell Viability Assay (Promega).At 8 h after the addition of the drug, cells treated with ceruleninexhibited reduced viability. This effect was more prominent after24 h. However, treatment with TOFA did not decrease cell viability(Fig. 1A, top and lower panels).Secondly, to study the inhibitory effect of these drugs on ZIKVreplication, A549 cells were infected with a generic Asian strain-derived ZIKV expressing nanoluciferase (Nluc) followed by foot-and-mouth disease virus 2A autoprotease, ubiquitin sequence(Fig. 1B); these elements are required to liberate Nluc fromduplicated capsid protein C. The cells were infected at amultiplicity of infection (MOI) of 0.1 or 1 and the drugs appliedat either 4 or 16 h post-infection (hpi), respectively. Due to thetoxicity of cerulenin, cells were lysed 8 h post-treatment and Nlucle under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Fig. 1. (A) A549 cells were treated with different concentrations of drugs for 8 or 24 h. Dimethyl sulphoxide (DMSO) was used as a negative control and cell viability wasmeasured using a CellTitre-Glo Luminescent Cell Viability Assay kit. The experiment was repeated independently three times. (B) Representation of reporter viruses: (1) Zikavirus expressing nanoluciferase (ZIKV-NlucUbi); and (2) Semliki Forest virus expressing ﬁreﬂy luciferase (SFV4(3H)-FFLuc). (C) A549 cells were infected with SFV4(3H)-FFLucat a multiplicity of infection (MOI) of 1 and drugs were added at 1 h post-infection (hpi). After 8 hpi, the cells were lysed and ﬁreﬂy luciferase (FFLuc) activity was measured.(D) A549 cells were infected with ZIKV-NLucUbi at MOIs of 0.1 or 1 and drugs were added at 1, 4 or 16 h. Cells were lysed after either 12 or 24 hpi and nanoluciferase (Nluc)activity was measured. Experiments in (C) and (D) were conducted independently twice in quadruplicate; the relative mean values with standard errors are depicted. (E)A549 cells were infected with Brazilian ZIKV strain PE243 at a MOI of 1. Drugs were added either 1 or 4 hpi; total RNA was isolated at 12 or 24 hpi, respectively. Genomic RNAlevels were analysed by reverse transcriptase quantitative PCR (RT-qPCR) using cellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal control. Theexperiment was repeated independently three times and the relative mean values with standard error are depicted. *P < 0.01 in Student’s t test.J. Royle et al. / The Veterinary Journal 230 (2017) 62–64 6364 J. Royle et al. / The Veterinary Journal 230 (2017) 62–64activity was measured using the Nano-Glo kit (Promega). We alsocompared ZIKV to a recombinant strain of SFV expressing cleavableﬁreﬂy luciferase (FFLuc; designated SFV4(3H)-FFLuc) (Fig. 1B).A549 cells were infected at a MOI of 1 and drugs were added 1 hpi.As expected, both cerulenin and TOFA inhibited SFV; this effectwas concentration dependent, since higher concentrationsresulted in reduced luciferase amounts (Fig. 1C). Similar effectshave been shown previously for CHIKV (Karlas et al., 2016), a closerelative of SFV. The results of TOFA treatment on ZIKV showed that,if Nluc levels were measured 12 hpi (Fig. 1D, MOI 1, drugs added at4 hpi) at the highest drug concentration, only a 20% reduction inNluc levels was observed, thus indicating a weak decrease in ZIKVreplication. This weak effect was also present when cells werelysed at 24 hpi (Fig. 1D, MOI 0.1, drugs added at 16 hpi). However,regardless of the concentration of cerulenin used, there was nodecrease in Nluc levels. This suggested there was no effect of thisdrug on ZIKV replication.These results were unexpected, since the dependence ofﬂaviviruses on lipid biosynthesis is well documented. We thereforeperformed a further experiment, whereby A549 cells were infectedat a MOI of 1, with drugs added at 1 hpi and Nluc activity measuredat 24 hpi. TOFA was again found to have antiviral effects only at thehighest concentration, resulting in a weak, non-signiﬁcant, 20%reduction in luciferase (Fig. 1D). However, despite its observedtoxicity, cerulenin did not decrease virus replication.To conﬁrm these ﬁndings, cells were infected with the wild-type Brazilian strain ZIKV PE243 (Donald et al., 2016) at an MOI of 1.Cells were then treated with both drugs either at 1 or 4 hpi andtotal RNA was collected at 12 or 24 hpi. Using reverse transcriptase(RT) quantitative PCR (RT-qPCR), we found that neither ceruleninnor TOFA decreased virus genomic RNA levels signiﬁcantly (exceptfor a weak reduction at 12 hpi with cerulenin), thereby suggestingthat neither drug affects virus replication (Fig. 1E). The resistanceof ZIKV in A549 cells under the conditions tested here wasunexpected, since dependence on ACC and FASN has been shownfor different ﬂaviviruses including DENV and hepatitis C virus(HCV) (Yang et al., 2008; Heaton et al., 2010). However, both DENVand HCV infections are known to increase production of fatty acidsand higher levels of FASN expression.It is therefore possible that heightened production of FASNcould occur in ZIKV infected cells, which may result in relativeinsensitivity to drugs. Both drugs had the expected antiviral effectsagainst SFV infection and targeting fatty acids synthesis may beone way to treat alphavirus infections. However, a more detailedanalysis of the metabolic activities in cells associated with ZIKV isrequired.Conﬂict of interest statementNone of the authors of this paper has a ﬁnancial or personalrelationship with other people or organisations that couldinappropriately inﬂuence or bias the content of the paper.AcknowledgmentThis work was funded by UK Medical Research Council(MC_UU_12014, MR/N017552/1) (AK).ReferencesDonald, C.L., Brennan, B., Cumberworth, S.L., Rezelj, V.V., Clark, J.J., Cordeiro, M.T.,Freitas de Oliveira Franca, R., Pena, L.J., Wilkie, G.S., Da Silva Filipe, A., et al., 2016.Full genome sequence and sfRNA interferon antagonist activity of Zika virusfrom Recife, Brazil. PLoS Neglected Tropical Diseases 10, e0005048.Ferreira-de-Brito, A., Ribeiro, I.P., Miranda, R.M., Fernandes, R.S., Campos, S.S., Silva,K.A., Castro, M.G., Bonaldo, M.C., Brasil, P., Lourenco-de-Oliveira, R., 2016. Firstdetection of natural infection of Aedes aegypti with Zika virus in Brazil andthroughout South America. Memórias do Instituto Oswaldo Cruz 111, 655–658.Heaton, N.S., Perera, R., Berger, K.L., Khadka, S., Lacount, D.J., Kuhn, R.J., Randall, G.,2010. Dengue virus nonstructural protein 3 redistributes fatty acid synthase tosites of viral replication and increases cellular fatty acid synthesis. Proceedingsof the National Academy of Sciences of the United States of America 107, 17345–17350.Karlas, A., Berre, S., Couderc, T., Varjak, M., Braun, P., Meyer, M., Gangneux, N., Karo-Astover, L., Weege, F., Raftery, M., et al., 2016. A human genome-wide loss-of-function screen identiﬁes effective chikungunya antiviral drugs. NatureCommunications 7, 11320.Martin-Acebes, M.A., Vazquez-Calvo, A., Saiz, J.C., 2016. Lipids and ﬂaviviruses,present and future perspectives for the control of dengue, Zika, and West Nileviruses. Progress in Lipid Research 64, 123–137.Russo, F.B., Jungmann, P., Beltrao-Braga, P.C., 2017. Zika infection and thedevelopment of neurological defects. Cellular Microbiology 19.Weaver, S.C., Reisen, W.K., 2010. Present and future arboviral threats. AntiviralResearch 85, 328–345.Yang, W., Hood, B.L., Chadwick, S.L., Liu, S., Watkins, S.C., Luo, G., Conrads, T.P., Wang,T., 2008. Fatty acid synthase is up-regulated during hepatitis C virus infectionand regulates hepatitis C virus entry and production. Hepatology 48, 1396–1403.

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Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses

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