Publications by authors named "Thomas Hoenen"

72 Publications

iPSC screening for drug repurposing identifies anti-RNA virus agents modulating host cell susceptibility.

FEBS Open Bio 2021 05 6;11(5):1452-1464. Epub 2021 Apr 6.

Center for iPS Cell Research and Application (CiRA), Kyoto University, Japan.

Human pathogenic RNA viruses are threats to public health because they are prone to escaping the human immune system through mutations of genomic RNA, thereby causing local outbreaks and global pandemics of emerging or re-emerging viral diseases. While specific therapeutics and vaccines are being developed, a broad-spectrum therapeutic agent for RNA viruses would be beneficial for targeting newly emerging and mutated RNA viruses. In this study, we conducted a screen of repurposed drugs using Sendai virus (an RNA virus of the family Paramyxoviridae), with human-induced pluripotent stem cells (iPSCs) to explore existing drugs that may present anti-RNA viral activity. Selected hit compounds were evaluated for their efficacy against two important human pathogens: Ebola virus (EBOV) using Huh7 cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using Vero E6 cells. Selective estrogen receptor modulators (SERMs), including raloxifene, exhibited antiviral activities against EBOV and SARS-CoV-2. Pioglitazone, a PPARγ agonist, also exhibited antiviral activities against SARS-CoV-2, and both raloxifene and pioglitazone presented a synergistic antiviral effect. Finally, we demonstrated that SERMs blocked entry steps of SARS-CoV-2 into host cells. These findings suggest that the identified FDA-approved drugs can modulate host cell susceptibility against RNA viruses.
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http://dx.doi.org/10.1002/2211-5463.13153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091584PMC
May 2021

Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus.

Microorganisms 2020 Aug 11;8(8). Epub 2020 Aug 11.

Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany.

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.
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http://dx.doi.org/10.3390/microorganisms8081215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463789PMC
August 2020

Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35.

J Virol 2020 07 30;94(16). Epub 2020 Jul 30.

Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain. Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.
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http://dx.doi.org/10.1128/JVI.02100-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394894PMC
July 2020

The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription.

Cells 2020 05 1;9(5). Epub 2020 May 1.

Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany.

Ebola virus (EBOV) is a zoonotic pathogen causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and extent of outbreaks has increased, highlighting the importance of better understanding the molecular aspects of EBOV infection and host cell interactions to control this virus more efficiently. Many viruses, including EBOV, have been shown to recruit host proteins for different viral processes. Based on a genome-wide siRNA screen, we recently identified the cellular host factor carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being involved in EBOV RNA synthesis. However, mechanistic details of how this host factor plays a role in the EBOV life cycle remain elusive. In this study, we analyzed the functional and molecular interactions between EBOV and CAD. To this end, we used siRNA knockdowns in combination with various reverse genetics-based life cycle modelling systems and additionally performed co-immunoprecipitation and co-immunofluorescence assays to investigate the influence of CAD on individual aspects of the EBOV life cycle and to characterize the interactions of CAD with viral proteins. Following this approach, we could demonstrate that CAD directly interacts with the EBOV nucleoprotein NP, and that NP is sufficient to recruit CAD into inclusion bodies dependent on the glutaminase (GLN) domain of CAD. Further, siRNA knockdown experiments indicated that CAD is important for both viral genome replication and transcription, while substrate rescue experiments showed that the function of CAD in pyrimidine synthesis is indeed required for those processes. Together, this suggests that NP recruits CAD into inclusion bodies via its GLN domain in order to provide pyrimidines for EBOV genome replication and transcription. These results define a novel mechanism by which EBOV hijacks host cell pathways in order to facilitate genome replication and transcription and provide a further basis for the development of host-directed broad-spectrum antivirals.
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http://dx.doi.org/10.3390/cells9051126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290923PMC
May 2020

Antiviral activity of sertindole, raloxifene and ibutamoren against transcription and replication-competent Ebola virus-like particles.

BMB Rep 2020 Mar;53(3):166-171

Virus Research Group, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Korea.

A chemical library comprising 2,354 drug-like compounds was screened using a transcription and replication-competent viruslike particle (trVLP) system implementing the whole Ebola virus (EBOV) life cycle. Dose-dependent inhibition of Ebola trVLP replication was induced by 15 hit compounds, which primarily target different types of G protein-coupled receptors (GPCRs). Based on the chemical structure, the compounds were divided into three groups, diphenylmethane derivatives, promazine derivatives and chemicals with no conserved skeletons. The third group included sertindole, raloxifene, and ibutamoren showing prominent antiviral effects in cells. They downregulated the expression of viral proteins, including the VP40 matrix protein and the envelope glycoprotein. They also reduced the amount of EBOV-derived tetracistronic minigenome RNA incorporated into progeny trVLPs in the culture supernatant. Particularly, ibutamoren, which is a known agonist of growth hormone secretagogue receptor (GHSR), showed the most promising antiviral activity with a 50% effective concentration of 0.2 μM, a 50% cytotoxic concentration of 42.4 μM, and a selectivity index of 222.8. Here, we suggest a strategy for development of anti-EBOV therapeutics by adopting GHSR agonists as hit compounds. [BMB Reports 2020; 53(3): 166-171].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118351PMC
March 2020

The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression.

Cells 2020 01 11;9(1). Epub 2020 Jan 11.

Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald, Germany.

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.
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http://dx.doi.org/10.3390/cells9010187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017048PMC
January 2020

High-throughput screening for negative-stranded hemorrhagic fever viruses using reverse genetics.

Antiviral Res 2019 10 26;170:104569. Epub 2019 Jul 26.

Junior Research Group - Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald, Insel Riems, Germany. Electronic address:

Viral hemorrhagic fevers (VHFs) cause thousands of fatalities every year, but the treatment options for their management remain very limited. In particular, the development of therapeutic interventions is restricted by the lack of commercial viability of drugs targeting individual VHF agents. This makes approaches like drug repurposing and/or the identification of broad range therapies (i.e. those directed at host responses or common proviral factors) highly attractive. However, the identification of candidates for such antiviral repurposing or of host factors/pathways important for the virus life cycle is reliant on high-throughput screening (HTS). Recently, such screening work has been increasingly facilitated by the availability of reverse genetics-based approaches, including tools such as full-length clone (FLC) systems to generate reporter-expressing viruses or various life cycle modelling (LCM) systems, many of which have been developed and/or greatly improved during the last years. In particular, since LCM systems are capable of modelling specific steps in the life cycle, they are a valuable tool for both targeted screening (i.e. for inhibitors of a specific pathway) and mechanism of action studies. This review seeks to summarize the currently available reverse genetics systems for negative-sense VHF causing viruses (i.e. arenaviruses, bunyaviruses and filoviruses), and to highlight the recent advancements made in applying these systems for HTS to identify either antivirals or new virus-host interactions that might hold promise for the development of future treatments for the infections caused by these deadly but neglected virus groups.
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http://dx.doi.org/10.1016/j.antiviral.2019.104569DOI Listing
October 2019

Therapeutic strategies to target the Ebola virus life cycle.

Nat Rev Microbiol 2019 10 24;17(10):593-606. Epub 2019 Jul 24.

Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA.

Following the Ebola virus disease epidemic in west Africa, there has been increased awareness of the need for improved therapies for emerging diseases, including viral haemorrhagic fevers such as those caused by Ebola virus and other filoviruses. Our continually improving understanding of the virus life cycle coupled with the increased availability of 'omics' analyses and high-throughput screening technologies has enhanced our ability to identify potential viral and host factors and aspects involved in the infection process that might be intervention targets. In this Review we address compounds that have shown promise to various degrees in interfering with the filovirus life cycle, including monoclonal antibodies such as ZMapp, mAb114 and REGN-EB3 and inhibitors of viral RNA synthesis such as remdesivir and TKM-Ebola. Furthermore, we discuss the general potential of targeting aspects of the virus life cycle such as the entry process, viral RNA synthesis and gene expression, as well as morphogenesis and budding.
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http://dx.doi.org/10.1038/s41579-019-0233-2DOI Listing
October 2019

Sequencing of mRNA from Whole Blood using Nanopore Sequencing.

J Vis Exp 2019 06 3(148). Epub 2019 Jun 3.

Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut;

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.
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http://dx.doi.org/10.3791/59377DOI Listing
June 2019

Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins.

J Gen Virol 2019 05 24;100(5):760-772. Epub 2019 Apr 24.

1 Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany.

Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein-protein interactions on a functional level.
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http://dx.doi.org/10.1099/jgv.0.001261DOI Listing
May 2019

Calu-3 cells are largely resistant to entry driven by filovirus glycoproteins and the entry defect can be rescued by directed expression of DC-SIGN or cathepsin L.

Virology 2019 06 3;532:22-29. Epub 2019 Apr 3.

Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany; Faculty of Biology and Psychology, Wilhelm-Weber-Str. 2, University Göttingen, 37073 Göttingen, Germany. Electronic address:

Priming of the viral glycoprotein (GP) by the cellular proteases cathepsin B and L (CatB, CatL) is believed to be essential for cell entry of filoviruses. However, pseudotyping systems that predominantly produce non-filamentous particles have frequently been used to prove this concept. Here, we report that GP-mediated entry of retroviral-, rhabdoviral and filoviral particles depends on CatB/CatL activity and that this effect is cell line-independent. Moreover, we show that the human cell line Calu-3, which expresses low amounts of CatL, is largely resistant to entry driven by diverse filovirus GPs. Finally, we demonstrate that Calu-3 cell entry mediated by certain filovirus GPs can be rescued upon directed expression of CatL or DC-SIGN. Our results identify Calu-3 cells as largely resistant to filovirus GP-driven entry and demonstrate that entry is limited at the stage of virion attachment and GP priming.
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http://dx.doi.org/10.1016/j.virol.2019.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112014PMC
June 2019

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies.

Antiviral Res 2019 03 19;163:106-116. Epub 2019 Jan 19.

Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald, Insel Riems, Germany; Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, MT, USA. Electronic address:

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species.
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http://dx.doi.org/10.1016/j.antiviral.2019.01.006DOI Listing
March 2019

Tetherin Inhibits Nipah Virus but Not Ebola Virus Replication in Fruit Bat Cells.

J Virol 2019 02 17;93(3). Epub 2019 Jan 17.

Infection Biology Unit, German Primate Center, Göttingen, Germany

Ebola virus (EBOV) and Nipah virus (NiV) infection of humans can cause fatal disease and constitutes a public health threat. In contrast, EBOV and NiV infection of fruit bats, the putative (EBOV) or proven (NiV) natural reservoir, is not associated with disease, and it is currently unknown how these animals control the virus. The human interferon (IFN)-stimulated antiviral effector protein tetherin (CD317, BST-2) blocks release of EBOV- and NiV-like particles from cells and is counteracted by the EBOV glycoprotein (GP). In contrast, it is unknown whether fruit bat tetherin restricts virus infection and is susceptible to GP-driven antagonism. Here, we report the sequence of fruit bat tetherin and show that its expression is IFN stimulated and associated with strong antiviral activity. Moreover, we demonstrate that EBOV-GP antagonizes tetherin orthologues of diverse species but fails to efficiently counteract fruit bat tetherin in virus-like particle (VLP) release assays. However, unexpectedly, tetherin was dispensable for robust IFN-mediated inhibition of EBOV spread in fruit bat cells. Thus, the VLP-based model systems mimicking tetherin-mediated inhibition of EBOV release and its counteraction by GP seem not to adequately reflect all aspects of EBOV release from IFN-stimulated fruit bat cells, potentially due to differences in tetherin expression levels that could not be resolved by the present study. In contrast, tetherin expression was essential for IFN-dependent inhibition of NiV infection, demonstrating that IFN-induced fruit bat tetherin exerts antiviral activity and may critically contribute to control of NiV and potentially other highly virulent viruses in infected animals. Ebola virus and Nipah virus (EBOV and NiV) can cause fatal disease in humans. In contrast, infected fruit bats do not develop symptoms but can transmit the virus to humans. Why fruit bats but not humans control infection is largely unknown. Tetherin is an antiviral host cell protein and is counteracted by the EBOV glycoprotein in human cells. Here, employing model systems, we show that tetherin of fruit bats displays higher antiviral activity than human tetherin and is largely resistant against counteraction by the Ebola virus glycoprotein. Moreover, we demonstrate that induction of tetherin expression is critical for interferon-mediated inhibition of NiV but, for at present unknown reasons, not EBOV spread in fruit bat cells. Collectively, our findings identify tetherin as an antiviral effector of innate immune responses in fruit bats, which might allow these animals to control infection with NiV and potentially other viruses that cause severe disease in humans.
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http://dx.doi.org/10.1128/JVI.01821-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340021PMC
February 2019

High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system.

Antiviral Res 2018 10 24;158:226-237. Epub 2018 Aug 24.

Applied Molecular Virology Laboratory, Institut Pasteur Korea, South Korea. Electronic address:

The massive epidemic of Ebola virus disease (EVD) in West Africa, followed in recent months by two outbreaks in the Democratic Republic of the Congo, underline the importance of this severe disease. Because Ebola virus (EBOV) must be manipulated under biosafety level 4 (BSL4) containment, the discovery and development of virus-specific therapies have been hampered. Recently, a transient transfection-based transcription- and replication competent virus-like particle (trVLP) system was described, enabling modeling of the entire EBOV life cycle under BSL2 conditions. Using this system, we optimized the condition for bulk co-transfection of multiple plasmids, developed a luciferase reporter-based assay in 384-well microtiter plates, and performed a high-throughput screening (HTS) campaign of an 8,354-compound collection consisting of U.S. Food & Drug Administration (FDA) -approved drugs, bioactives, kinase inhibitors, and natural products in duplicates. The HTS achieved a good signal-to-background ratio with a low percent coefficient of variation resulting in Z' = 0.7, and data points were reproducible with R = 0.89, indicative of a robust assay. After applying stringent hit selection criteria of ≥70% EBOV trVLP inhibition and ≥70% cell viability, 381 hits were selected targeting early, entry, and replication steps and 49 hits targeting late, maturation, and secretion steps in the viral life cycle. Of the total 430 hits, 220 were confirmed by dose-response analysis in the primary HTS assay. They were subsequently triaged by time-of-addition assays, then clustered and ranked according to their chemical structures, biological functions, therapeutic index, and maximum inhibition. Several novel drugs have been identified to very efficiently inhibit EBOV. Interestingly, most showed pharmacological activity in treatments for central nervous system-related diseases. We developed and screened an HTS assay using the novel EBOV trVLP system. Newly identified inhibitors are useful tools to study the poorly understood EBOV life cycle. In addition, they also provide opportunities to either repurpose FDA-approved drugs or develop novel viral interventions to combat EVD.
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http://dx.doi.org/10.1016/j.antiviral.2018.08.013DOI Listing
October 2018

A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle.

Genome Med 2018 08 7;10(1):58. Epub 2018 Aug 7.

Laboratory of Virology, Division of Intramural Research, National Institute for Allergy and Infectious Diseases, National Institutes of Health, 903 S 4th St., Hamilton, MT, 59840, USA.

Background: The 2014-2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4).

Methods: In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory.

Results: Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses.

Conclusions: This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitro, and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.
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http://dx.doi.org/10.1186/s13073-018-0570-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090742PMC
August 2018

Generation and Optimization of a Green Fluorescent Protein-Expressing Transcription and Replication-Competent Virus-Like Particle System for Ebola Virus.

J Infect Dis 2018 11;218(suppl_5):S360-S364

Institute for Molecular Virology and Cell Biology.

Work with infectious Ebola virus is restricted to biosafety level (BSL) 4 laboratories. To overcome this limitation, life cycle modeling systems, which recapitulate part or all of the virus life cycle under BSL-1 or -2 conditions, have been developed. The tetracistronic transcription and replication-competent virus-like particle (trVLP) system is currently the most advanced of these systems and is particularly useful for drug screening. However, previous versions have used luciferase reporters, limiting the types of screening assays that can be performed. Here we describe the generation and optimization of a green fluorescent protein-expressing tetracistronic trVLP system, enabling high-content imaging and flow cytometry approaches.Summary: Transcription and replication-competent virus-like particle (trVLP) systems are powerful tools to model the life cycle of highly pathogenic Ebola viruses. Here we describe the generation of a novel, GFP-based trVLP system that allows high content imaging and flow cytometry approaches.
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http://dx.doi.org/10.1093/infdis/jiy405DOI Listing
November 2018

Lifecycle modelling systems support inosine monophosphate dehydrogenase (IMPDH) as a pro-viral factor and antiviral target for New World arenaviruses.

Antiviral Res 2018 09 19;157:140-150. Epub 2018 Jul 19.

Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, MT, USA; Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald, Insel Riems, Germany. Electronic address:

Infection with Junín virus (JUNV) is currently being effectively managed in the endemic region using a combination of targeted vaccination and plasma therapy. However, the long-term sustainability of plasma therapy is unclear and similar resources are not available for other New World arenaviruses. As a result, there has been renewed interest regarding the potential of drug-based therapies. To facilitate work on this issue, we present the establishment and subsequent optimization of a JUNV minigenome system to a degree suitable for high-throughput miniaturization, thereby providing a screening platform focused solely on factors affecting RNA synthesis. Using this tool, we conducted a limited drug library screen and identified AVN-944, a non-competitive inosine monophosphate dehydrogenase (IMPDH) inhibitor, as an inhibitor of arenavirus RNA synthesis. We further developed a transcription and replication competent virus-like particle (trVLP) system based on these minigenomes and used it to screen siRNAs against IMPDH, verifying its role in supporting arenavirus RNA synthesis. The antiviral effect of AVN-944, as well as siRNA inhibition, on JUNV RNA synthesis supports that, despite playing only a minor role in the activity of ribavirin, exclusive IMPDH inhibitors may indeed have significant therapeutic potential for use against New World arenaviruses. Finally, we confirmed that AVN-944 is also active against arenavirus infection in cell culture, supporting the suitability of arenavirus lifecycle modelling systems as tools for the screening and identification, as well as the mechanistic characterization, of novel antiviral compounds.
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http://dx.doi.org/10.1016/j.antiviral.2018.07.009DOI Listing
September 2018

Serological Evidence for the Circulation of Ebolaviruses in Pigs From Sierra Leone.

J Infect Dis 2018 11;218(suppl_5):S305-S311

Friedrich-Loeffler-Institut Institute of Novel and Emerging Infectious Diseases, Greifswald - Insel Riems, Germany.

Many human ebolavirus outbreaks have been linked to contact with wildlife including nonhuman primates and bats, which are assumed to serve as host species. However, it is largely unknown to what extent other animal species, particularly livestock, are involved in the transmission cycle or act as additional hosts for filoviruses. Pigs were identified as a susceptible host for Reston virus with subsequent transmission to humans reported in the Philippines. To date, there is no evidence of natural Ebola virus (EBOV) infection in pigs, although pigs were shown to be susceptible to EBOV infection under experimental settings. To investigate the potential role of pigs in the ecology of EBOV, we analyzed 400 porcine serum samples from Sierra Leone for the presence of ebolavirus-specific antibodies. Three samples reacted with ebolavirus nucleoproteins but had no neutralizing antibodies. Our results (1) suggest the circulation of ebolaviruses in swine in Sierra Leone that are antigenically related but not identical to EBOV and (2) could represent undiscovered ebolaviruses with unknown pathogenic and/or zoonotic potential.
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http://dx.doi.org/10.1093/infdis/jiy330DOI Listing
November 2018

Analysis of a Putative Late Domain Using an Ebola Virus Transcription and Replication-Competent Virus-Like Particle System.

J Infect Dis 2018 11;218(suppl_5):S355-S359

Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

The Ebola virus (EBOV) matrix protein VP40 drives budding of virions and encodes 2 overlapping late domain motifs at amino acid positions 7-13 (PTAPPEY). However, these motifs are not absolutely essential for replication in cell culture, and recently a potential third late domain motif (YPx(6)I) was proposed at amino acid positions 18-26 of VP40. To analyze the importance of this motif in viral budding, we used a transcription and replication-competent virus-like particle system. Using this system, we show that this motif does not contribute to EBOV budding or particle propagation.
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http://dx.doi.org/10.1093/infdis/jiy247DOI Listing
November 2018

Characterization of the catalytic center of the Ebola virus L polymerase.

PLoS Negl Trop Dis 2017 Oct 9;11(10):e0005996. Epub 2017 Oct 9.

Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

Background: Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized.

Methodology/principal Findings: Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.

Conclusions/significance: We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle.
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http://dx.doi.org/10.1371/journal.pntd.0005996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648267PMC
October 2017

Minigenome Systems for Filoviruses.

Authors:
Thomas Hoenen

Methods Mol Biol 2018 ;1604:237-245

Friedrich-Loeffler-Institut, Institute for Molecular Virology and Cell Biology, Greifswald-Isle of Riems, Germany.

Filoviruses are among the most pathogenic viruses known to man, and work with live viruses is restricted to maximum containment laboratories. In order to study individual aspects of the virus life cycle outside of maximum containment laboratories, life cycle modeling systems have been established, which use reporter-encoding miniature versions of the viral genome called minigenomes. With basic minigenome systems viral genome replication and transcription can be studied, whereas more advanced systems also allow us to model other aspects of the virus life cycle outside of a maximum containment laboratory. These systems, therefore, represent powerful tools to study the biology of filoviruses, and for the screening and development of antivirals.
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http://dx.doi.org/10.1007/978-1-4939-6981-4_18DOI Listing
June 2018

Reverse Genetics of Filoviruses.

Curr Top Microbiol Immunol 2017;411:421-445

Integrated Research Facility at Fort Detrick (IRF-Frederick), Division of Clinical Research (DCR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), B-8200 Research Plaza, Fort Detrick, Frederick, MD, 21702, USA.

Reverse genetics systems are used for the generation of recombinant viruses. For filoviruses, this technology has been available for more than 15 years and has been used to investigate questions regarding the molecular biology, pathogenicity, and host adaptation determinants of these viruses. Further, reporter-expressing, recombinant viruses are increasingly used as tools for screening for and characterization of candidate medical countermeasures. Thus, reverse genetics systems represent powerful research tools. Here we provide an overview of available reverse genetics systems for the generation of recombinant filoviruses, potential applications, and the achievements that have been made using these systems.
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http://dx.doi.org/10.1007/82_2017_55DOI Listing
May 2019

Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging.

Sci Rep 2017 08 9;7(1):7698. Epub 2017 Aug 9.

Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, 59840, USA.

Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.
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http://dx.doi.org/10.1038/s41598-017-08167-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550494PMC
August 2017

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

PLoS One 2017 26;12(6):e0179728. Epub 2017 Jun 26.

Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484481PMC
September 2017

Luciferase-Expressing Ebolaviruses as Tools for Screening of Antivirals.

Authors:
Thomas Hoenen

Methods Mol Biol 2017 ;1628:189-194

Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany.

Ebolaviruses cause severe hemorrhagic fever with high case fatality rates. Despite recent progress, there is a continued need for the development of antivirals against these viruses. Reporter-expressing ebolaviruses, which can be generated using reverse genetics systems, are powerful tools for antiviral screening. While viruses expressing fluorescent reporters are amenable for this purpose and can be used for high-content imaging-type screens, as an alternative, luciferase-expressing reporter viruses have recently been developed and have the advantages of being extremely easy to use and having short assay times. Here we provide a detailed protocol for the use of such a luciferase-expressing reporter virus for antiviral screening in a 96-well format, with parallel assessment of cytotoxicity of the screened compounds.
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http://dx.doi.org/10.1007/978-1-4939-7116-9_14DOI Listing
March 2018

Modeling the Ebolavirus Life Cycle with Transcription and Replication-Competent Viruslike Particle Assays.

Methods Mol Biol 2017 ;1628:119-131

Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany.

Ebolaviruses are the causative agent of a severe hemorrhagic fever with high case fatality rates, for which no approved specific therapy is available. As biosafety level 4 (BSL4) agents, work with live ebolaviruses is restricted to maximum containment laboratories. Transcription and replication-competent viruslike particle (trVLP) systems are reverse genetics-based life cycle modeling systems that allow researchers to model virtually the entire ebolavirus life cycle outside of a maximum containment laboratory. These systems can be used to dissect the virus life cycle, and thus increase our understanding of virus biology, as well as for more applied uses such as the screening and development of novel antivirals, and thus represent powerful tools for work on ebolaviruses.
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http://dx.doi.org/10.1007/978-1-4939-7116-9_9DOI Listing
March 2018

Forty Years of Ebolavirus Molecular Biology: Understanding a Novel Disease Agent Through the Development and Application of New Technologies.

Methods Mol Biol 2017 ;1628:15-38

Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany.

Molecular biology is a broad discipline that seeks to understand biological phenomena at a molecular level, and achieves this through the study of DNA, RNA, proteins, and/or other macromolecules (e.g., those involved in the modification of these substrates). Consequently, it relies on the availability of a wide variety of methods that deal with the collection, preservation, inactivation, separation, manipulation, imaging, and analysis of these molecules. As such the state of the art in the field of ebolavirus molecular biology research (and that of all other viruses) is largely intertwined with, if not driven by, advancements in the technical methodologies available for these kinds of studies. Here we review of the current state of our knowledge regarding ebolavirus biology and emphasize the associated methods that made these discoveries possible.
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http://dx.doi.org/10.1007/978-1-4939-7116-9_2DOI Listing
March 2018

Reverse Genetics Systems for Filoviruses.

Methods Mol Biol 2017 ;1602:159-170

Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA.

Filoviruses are among the most pathogenic viruses known to man. Reverse genetics systems, in particular full-length clone systems, allow the generation of recombinant filoviruses, which can be used to study virus biology, but also for applied uses such as screening for countermeasures. Here we describe the generation of recombinant filoviruses from cDNA.
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http://dx.doi.org/10.1007/978-1-4939-6964-7_11DOI Listing
March 2018

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses.

J Virol 2017 08 12;91(15). Epub 2017 Jul 12.

Institute of Virology, Medical Center, University of Freiburg, Freiburg, Germany

Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses.
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http://dx.doi.org/10.1128/JVI.00361-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512242PMC
August 2017

The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection.

PLoS Negl Trop Dis 2017 04 12;11(4):e0005540. Epub 2017 Apr 12.

Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America.

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.
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http://dx.doi.org/10.1371/journal.pntd.0005540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402990PMC
April 2017