Abstract
Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.
Highlights
Filoviruses (Mononegavirales: Filoviridae) have linear non-segmented negative-senseRNA genomes, consisting of the canonical genes 3 -NP-VP35-VP40-GPVP30-VP24-L-5 that encode nucleoprotein (NP), polymerase cofactor (VP35), matrix protein (VP40), spike glycoprotein (GP1,2), transcriptional activator (VP30), RNA complexassociated protein (VP24), and large protein (L, including an RNA-directed RNA polymerase [RdRp] activity), respectively [1]
Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 (EBOV), the etiologic agent of Ebola virus disease (EVD) [3], caused the two largest filovirus disease outbreaks on record: 28,652 cases with 11,323 deaths were reported during the 2013–2016 EVD outbreak in Western Africa; and 3481 cases with 2299 deaths occurred during a 2018–2020 EVD outbreak in the Democratic Republic of the Congo [4,5]
These data indicate that the VP40-based screening is suitable for th identification of novel EBOV medical countermeasure (MCM) and that sangivamycin could potentia3lolyf 20be devel oped as a broad-spectrum antiviral once its mechanism of action is further clarified
Summary
Filoviruses (Mononegavirales: Filoviridae) have linear non-segmented negative-sense. RNA genomes (up to 19.1 kb), consisting of the canonical genes 3 -NP-VP35-VP40-GPVP30-VP24-L-5 that encode nucleoprotein (NP), polymerase cofactor (VP35), matrix protein (VP40), spike glycoprotein (GP1,2), transcriptional activator (VP30), RNA complexassociated protein (VP24), and large protein (L, including an RNA-directed RNA polymerase [RdRp] activity), respectively [1]. Most small molecules that have proven highly efficacious in non-human primate models of EVD and/or have been evaluated in clinical trials are nucleoside analogs (e.g., favipiravir [15,16,17] and remdesivir [7]) These molecules are associated with a single mechanism of action, i.e., the inhibition of virus replication and/or transcription through interference with the L-contained RdRp activity [8,18,19]. Using an EBOV minigenome assay that does not encode or expres VP40, we demonstrate that these broad-spectrum effects are likely due to a highly efficien secondary interaction with RdRps or other viral proteins required for viral replication and/or transcription These data indicate that the VP40-based screening is suitable for th identification of novel EBOV MCMs and that sangivamycin could potentia3lolyf 20be devel oped as a broad-spectrum antiviral once its mechanism of action is further clarified. CtehlelsseanredsuCletlsl, CsaunlgtuivreamCyoncidnitiinohnisbited the replication of EBOV but, surprisingly, it inGhriibvietetd(CthhelorreopcleibcautsioanetohfioEpBsO) VV’esrcoloEse rceelalltsiv(eN, MR5A9R6V, ,BaEs IwRelelsaosutrhceesu,nMrelaanteadssas, VA LUaSssAa),vhiruums (aLnAeSmV)b,rcyoownpicoxkivdinruesy ((CHPEXKV)),29an3dT cvealclcsin(AiaTvCirCu,sM(VaAnCasVs)a,sw, VhiAch, UdoSAn)ot293T/T1 VescRexePecpl4oslr0sone,su(dwsBraVEceryePIds4Rei,0nmMetoseoroarntanuhscotartrclsiaooestgnases,st.wM,hUVaitastAhintnh,aRgeUsdsaseSRnaAbpsE,rs)BoVwoOaArdVe-,orsmUetphemiSencArtiagrvi)uen,imnrataoanlmeidnfpfeeerhoacdutstsesamaiatnyar3sent7hrlih°eakCqteeudplayioanredetdsodunc5efao%otrtorceCiavnnOichoroia2mgdlihenraleoyD(prHeleuifxcufilapbchtriei7eeocn)scnstcoe’lslsM(BodE aifnided/oEr atrgalnes’csriMpteiodni.uTmhe(sDe MdaEtaMin)d(iLcaiftee tTheacththneoVloPg4i0e-sb,aCseadrlsscbreaedn,inCgAis, sUuSitAab)lecofonrttahiening 10% ihdeeantt-ifiincaatcitoinvaotfendovfeeltEaBlObVoMviCnMe ssaenrdumthat(sFaBnSg)i.vaHmuycminacnoumldopnootecnyttiea-lldyebreivdeedvelmopaecdrophage a(Ms aDbMroasd) -wspeercetrduimffearnetnivtiaratel donfcreomits CmDec1h4a+nmisomnoofcayctteiosnanisdfucrutlhteurrceldaraifisepdr.eviously described [36,37]
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