Ebolavirus causes sporadic outbreaks of lethal hemorrhagic fever in humans with no currently approved therapy. Cells take up Ebolavirus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebolavirus entry into host cells requires the endosomal calcium channels called two pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs or small molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule we tested, inhibited infection of human macrophages, the primary target of Ebolavirus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebolavirus infection and may be effective targets for antiviral therapy.

Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.

BackgroundThe rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency.Methodology/Principal FindingsA large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo.Conclusions/SignificanceThe feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses.

A systematic screen of FDA-approved drugs was performed to identify compounds with in vitro antiviral activities against Ebola virus (EBOV). Compounds active (>50% viral inhibition and <30% cellular toxicity) at a single concentration were tested in dose-response assays to quantitate the antiviral activities in replication and viral entry assays as well as cytotoxicity in the Vero cell line used to conduct these assays. On the basis of the approved human dosing, toxicity/tolerability, and pharmacokinetic data, seven of these in vitro hits from different pharmacological classes (chloroquine (CQ), amiodarone, prochlorperazine, benztropine, azithromycin, chlortetracycline, and clomiphene) were evaluated for their in vivo efficacy at a single dose and were administered via either intraperitoneal (ip) or oral route. Initially, azithromycin (100 mg/kg, twice daily, ip), CQ (90 mg/kg, twice daily, ip), and amiodarone (60 mg/kg, twice daily, ip) demonstrated significant increases in survival in the mouse model. After repeat evaluation, only CQ was found to reproducibly give significant efficacy in the mouse model with this dosing regimen. Azithromycin and CQ were also tested in a guinea pig model of EBOV infection over a range of doses, but none of the doses increased survival, and drug-related toxicity was observed at lower doses than in the mouse. These results show the benefits and specific challenges associated with drug repurposing and highlight the need for careful evaluation of approved drugs as rapidly deployable countermeasures against future pandemics.

The phosphoinositide-3 kinase (PI3K) pathway regulates diverse cellular activities related to cell growth, migration, survival, and vesicular trafficking. It is known that Ebola virus requires endocytosis to establish an infection. However, the cellular signals that mediate this uptake were unknown for Ebola virus as well as many other viruses. Here, the involvement of PI3K in Ebola virus entry was studied. A novel and critical role of the PI3K signaling pathway was demonstrated in cell entry of Zaire Ebola virus (ZEBOV). Inhibitors of PI3K and Akt significantly reduced infection by ZEBOV at an early step during the replication cycle. Furthermore, phosphorylation of Akt-1 was induced shortly after exposure of cells to radiation-inactivated ZEBOV, indicating that the virus actively induces the PI3K pathway and that replication was not required for this induction. Subsequent use of pseudotyped Ebola virus and/or Ebola virus-like particles, in a novel virus entry assay, provided evidence that activity of PI3K/Akt is required at the virus entry step. Class 1A PI3Ks appear to play a predominant role in regulating ZEBOV entry, and Rac1 is a key downstream effector in this regulatory cascade. Confocal imaging of fluorescently labeled ZEBOV indicated that inhibition of PI3K, Akt, or Rac1 disrupted normal uptake of virus particles into cells and resulted in aberrant accumulation of virus into a cytosolic compartment that was non-permissive for membrane fusion. We conclude that PI3K-mediated signaling plays an important role in regulating vesicular trafficking of ZEBOV necessary for cell entry. Disruption of this signaling leads to inappropriate trafficking within the cell and a block in steps leading to membrane fusion. These findings extend our current understanding of Ebola virus entry mechanism and may help in devising useful new strategies for treatment of Ebola virus infection.