Several FDA-approved drugs, including bepridil and sertraline, may be therapeutics against filovirus infections.

Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)- and ex-US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.

Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC(50) 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann-Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

Ebolavirus (Ebov), an enveloped virus of the family Filoviridae , causes hemorrhagic fever in humans and nonhuman primates. The viral glycoprotein (GP) is solely responsible for virus–host membrane fusion, but how it does so remains elusive. Fusion occurs after virions reach an endosomal compartment where GP is proteolytically primed by cathepsins. Fusion by primed GP is governed by an internal fusion loop found in GP2, the fusion subunit. This fusion loop contains a stretch of hydrophobic residues, some of which have been shown to be critical for GP-mediated infection. Here we present liposome fusion data and NMR structures for a complete (54-residue) disulfide-bonded internal fusion loop (Ebov FL) in a membrane mimetic. The Ebov FL induced rapid fusion of liposomes of varying compositions at pH values at or below 5.5. Consistently, circular dichroism experiments indicated that the α-helical content of the Ebov FL in the presence of either lipid-mimetic micelles or small liposomes increases in samples exposed to pH ≤5.5. NMR structures in dodecylphosphocholine micelles at pH 7.0 and 5.5 revealed a conformational change from a relatively flat extended loop structure at pH 7.0 to a structure with an ∼90° bend at pH 5.5. Induction of the bend at low pH reorients and compacts the hydrophobic patch at the tip of the FL. We propose that these changes facilitate disruption of lipids at the site of virus–host cell membrane contact and, hence, initiate Ebov fusion.

Ebolavirus is an enveloped virus belonging to the Filoviridae family that causes hemorrhagic fever in humans. The mechanism of virus-host membrane fusion is not well characterized, but is mediated by the single viral glycoprotein, GP. Ebolavirus membrane fusion occurs from an endosomal compartment after proteolytic activation of GP by endosomal cathepsins and is governed by a patch of hydrophobic residues located within the internal fusion loop of the GP fusion subunit, GP2. Here we present liposome fusion data and NMR structures for the disulfide bound internal fusion loop (Ebov FL) at neutral and mildly acidic pH conditions.

ABSTRACT Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ∼10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.

ABSTRACT Most class I fusion proteins exist as trimers of dimers composed of a receptor binding and a fusion subunit. In their postfusion forms, the three fusion subunits form trimers of hairpins consisting of a central coiled coil (formed by the N-terminal helices), an intervening sequence, and a region containing the C helix (and flanking strands) that runs antiparallel to and packs in the grooves of the N-terminal coiled coil. For filoviruses and most retroviruses, the intervening sequence includes a “chain reversal region” consisting of a short stretch of hydrophobic residues, a Gly-Gly pair, a CX 6 CC motif, and a bulky hydrophobic residue. Maerz and coworkers (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74: 6614-6621, 2000) proposed a model for this region of human T-cell leukemia virus type 1 (HTLV-1) Env in which expulsion of the final bulky hydrophobic residue is important for early conformational changes and specific residues in the chain reversal region are important for forming the final, stable trimer of hairpins. Here, we used mutagenesis and pseudovirus entry assays to test this model for the avian retrovirus avian sarcoma/leukosis virus (ASLV) and the filovirus ebolavirus Zaire. Our results are generally consistent with the model proposed for HTLV-1 Env. In addition, we show with ASLV EnvA that the bulky hydrophobic residue following the CX 6 CC motif is required for the step of prehairpin target membrane insertion, whereas other residues are required for the foldback step of fusion. We further found that a His residue that is unique to the chain reversal region of ASLV EnvA controls the pH at which ASLV entry occurs.

Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).

Integrins are involved in the binding and internalization of both enveloped and nonenveloped viruses. By using 3 distinct cell systems—CHO cells lacking expression of α5β1-integrin, HeLa cells treated with siRNA to α5-integrin, and mouse β1-integrin knockout fibroblasts, we show that α5β1-integrin is required for efficient infection by pseudovirions bearing the ebolavirus glycoprotein (GP). These integrins are necessary for viral entry but not for binding or internalization. Given the need for endosomal cathepsins B and L (CatB and CatL) to prime GPs for fusion, we investigated the status of CatB and CatL in integrin-positive and integrin-negative cell lines. α5β1-Integrin-deficient cells lacked the double-chain (DC) forms of CatB and CatL, and this correlated with decreased CatL activity in integrin-negative CHO cells. These data indicate that α5β1-integrin-negative cells may be refractory to infection by GP pseudovirions because they lack the necessary priming machinery (the double-chain forms of CatB and CatL). In support of this model, we show that GP pseudovirions that have been preprimed in vitro to generate the 19-kDa form of GP overcome the requirement for α5β1-integrin for infection. These results provide further support for the requirement for endosomal cathepsins for ebolavirus infection, identify the DC forms of these cathepsins as previously unrecognized factors that contribute to cell tropism of this virus, and reveal a previously undescribed role for integrins during viral entry as regulators of endosomal cathepsins, which are required to prime the entry proteins of ebolavirus and other pathogenic viruses.

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

ABSTRACT To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since “bald” ΔG-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating.

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP(1,2)), a class I fusion protein whose structure was recently determined (J.E. Lee et al., Nature 454: 177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP(1) and GP(2) that remain after GP(1,2) is proteolytically primed by endosomal cathepsins for fusion and residues in GP(1) that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP(1,2) contains residues 33 to 190 of GP(1) and all residues of GP(2). The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP(1), by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP(1) residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP(1,2)-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP(1,2) is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP(1)) as a highly conserved region containing a two-stranded beta-sheet, the two intra-GP(1) disulfide bonds, and four critical Lys residues.

Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus.

ABSTRACT We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.

Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.

The receptor for avian sarcoma/leukosis virus subtype A (ASLV-A), Tva, is the simplest member of the low density lipoprotein receptor family containing a single ligand-binding repeat (LBR). Most LBRs contain a central Trp (Trp(33) in Tva) that is important for ligand binding and, for the low density lipoprotein receptor, is associated with familial hypercholesterolemia. The Tva ligand-binding module contains a second Trp (Trp(48)) that is part of a DEW motif present in a subset of LBRs. Trp(48) is important for ASLV-A infectivity. A soluble Tva (sTva) ligand-binding module is sufficient for ASLV-A infectivity. Tva interacts with the viral glycoprotein, and a soluble receptor-binding domain (SUA) binds sTva with picomolar affinity. We investigated whether Tva, a retroviral receptor, could behave as a classic LBR by assessing sTva interactions with the universal receptor-associated protein (RAP) and comparing these interactions with those between sTva and its viral ligand (SUA). To address the role of the two Trp residues in Tva function, we prepared sTva harboring mutations of Trp(33), Trp(48), or both and determined the binding kinetics with RAP and SUA. We found that sTva behaved as a "normal" receptor toward RAP, requiring both calcium and Trp(33) for binding. However, sTva binding to SUA required neither calcium nor Trp(33). Furthermore, sTva could bind both RAP and SUA simultaneously. These results show that the single LBR of Tva has two ligand-binding sites, raising the possibility that other LBRs may also.

ABSTRACT Using chemical inhibitors and small interfering RNA (siRNA), we have confirmed roles for cathepsin B (CatB) and cathepsin L (CatL) in Ebola virus glycoprotein (GP)-mediated infection. Treatment of Ebola virus GP pseudovirions with CatB and CatL converts GP1 from a 130-kDa to a 19-kDa species. Virus with 19-kDa GP1 displays significantly enhanced infection and is largely resistant to the effects of the CatB inhibitor and siRNA, but it still requires a low-pH-dependent endosomal/lysosomal function. These and other results support a model in which CatB and CatL prime GP by generating a 19-kDa intermediate that can be acted upon by an as yet unidentified endosomal/lysosomal enzyme to trigger fusion.

This chapter describes three assays to monitor receptor-induced association of the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus (ASLV) with target bilayers: (1) the original assay for monitoring binding of the EnvA ectodomain (EnvA-PI) to target membranes (liposomes), (2) a modified and miniaturized EnvA-PI-liposome binding assay, and (3) an assay to measure binding of intact sarcoma/leukosis virus subtype A (ASLV-A) virus particles to target membranes. These assays are also useful for studying other receptor-activated viral fusion proteins. When one viral glycoprotein and one “simple” host cell receptor are involved, it should be possible to develop assays directly analogous to those described above for studying Tva-induced binding of the EnvA ectodomain (EnvA-PI) to target membranes. A general prerequisite for a fusion protein/target membrane binding assay is a soluble and correctly oligomeric form of the viral fusion protein ectodomain. The simplest host cell receptors that would be amenable to this type of analysis are type I or type II integral membrane proteins. The soluble versions of the ectodomains of these receptors, produced by genetic engineering or proteolytic release, could then be used to trigger the cognate fusion protein. The methodology could, similarly, be applicable to multimembrane-spanning host cell receptors when the functional part of the receptor is tethered at only one end or where an ectodomain loop preserves enough structure to function as a soluble analog, perhaps by generating a cyclic peptide analog of the loop. The same “receptor reagents” could be employed for intact virus particle/target membrane binding assays.