Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak

Abstract

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. IMPORTANCEThe dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity.KEYWORDS West Africa, adaptive mutations, Ebola virus, glycoprotein, zoonotic infections E bola virus (EBOV) belongs to the family Filoviridae in the order Mononegavirales. The Ebola virus disease is characterized by severe fever accompanied by systemic inflammation and damage to the endothelial cell barrier leading to shock and multiorgan failure with high case/fatality rates (1). EBOV particles are filamentous (1 m in length, 80 nm in diameter) and composed of seven viral proteins. The nucleocapsid complex contains the viral RNA encapsidated by nucleoprotein NP and four additional viral proteins. The polymerase complex of VP35 and L is associated with NP via

Biochemical Basis for Increased Activity of Ebola Glycoprotein in the 2013–16 Epidemic

“…However, our studies do not support the suggestion that changes in residue 82 alter receptor affinity (Diehl et al, 2016; Dietzel et al, 2016; Urbanowicz et al, 2016). The recently obtained structure of GP bound to NPC1 revealed that the α-helix containing A82 is displaced by NPC1 binding, and it was proposed that this conformational change promotes the release of the internal fusion loop in GP2 (Wang et al, 2016).…”

“…This strongly predicts that V82 and I544 promote infection under conditions where expression of NPC1 and/or cathepsin B in target cells is low, such as in tissue monocytes and dendritic cells (DCs) in boundary tissues that are early targets for infection (Bray and Geisbert, 2005; Martinez et al, 2013). Expansion of infection in the host to cells where NPC1 and cathepsin B expression is low, as modeled by the use of 3.47 and CA074, may result in the reported increase in viremia associated with A82V (Diehl et al, 2016; Dietzel et al, 2016). A recent study found that Makona C07 containing T544 grows more slowly and is less virulent than 1976 Yambuku Ecran which, after cleavage, differs from Makona C07 solely by the presence of I544 in GP (Smither et al, 2016).…”

Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

“…Moreover, R111C substitution in NP has been proposed to be signi¯cant in NP oligomerization, NC formation and VP40 interaction, by comparative analysis of the amino acid sequences of ebola viral proteins from 2013 to 2015. 48 Alanine (A) to serine (S) substitutions, A233S and A279S, are close to the cleft of NP for RNA binding. The serine residues involved in these substitutions (A233S, A279S and S365H) are potential phosphorylation sites on the NP proteins, surrounding the cleft where NP binds to ssRNA (Figs.…”

“…48 It is also worth noting that changes in the conserved MTase region may add on to the increased pathogenicity (Table S2).…”

“…71 Several possible key di®erences on L have been proposed by comparative analysis between Zaire and Reston or other ebola species. 31,48,72 We mapped out the position of certain key residues: L283V, Y312F, A326S, T330D, S343Y, E350D, T361S, L365F in the binding core with VP35, and R654H, E689S, D759G in the Rdrp domain (listed in Table S2) on 3D model of its protein domain (Fig. 4).…”