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      Minimal In Vivo Efficacy of Iminosugars in a Lethal Ebola Virus Guinea Pig Model

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          Abstract

          The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin ( NB-DNJ) and N-(9-methoxynonyl)-1deoxynojirimycin (M ON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day M ON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. M ON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally-spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and M ON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars.

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          Most cited references45

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          Distribution and three-dimensional structure of AIDS virus envelope spikes.

          Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had approximately 14 and approximately 73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) 'head' of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein 'stalk' of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.
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            Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein.

            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.
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              Ebola virus VP24 binds karyopherin alpha1 and blocks STAT1 nuclear accumulation.

              Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-alpha/beta) and the ability of cells to respond to IFN-alpha/beta or IFN-gamma. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-alpha/beta production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-alpha/beta and IFN-gamma signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-alpha/beta and IFN-gamma signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin alpha1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin alpha2, alpha3, or alpha4. Overexpression of VP24 results in a loss of karyopherin alpha1-PY-STAT1 interaction, indicating that the VP24-karyopherin alpha1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 November 2016
                2016
                : 11
                : 11
                : e0167018
                Affiliations
                [1 ]Antiviral Research Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom
                [2 ]Public Health England, Porton Down, Salisbury, United Kingdom
                [3 ]The Royal Veterinary College, London, United Kingdom
                [4 ]Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, United Kingdom
                Division of Clinical Research, UNITED STATES
                Author notes

                Competing Interests: This work has been supported by Oxford Glycobiology Institute Endowment and a research grant from Emergent BioSolutions, Inc. (formerly Unither Virology LLC). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                • Conceptualization: JLM SGS SDD IT RH NZ.

                • Formal analysis: JLM SGS SDD DSA ACS RT GH.

                • Investigation: JLM SGS SDD IT AR DSA EMB EW RT.

                • Writing – original draft: JLM SGS.

                • Writing – review & editing: JLM SGS RAD NZ.

                Author information
                http://orcid.org/0000-0002-4383-4641
                Article
                PONE-D-16-34136
                10.1371/journal.pone.0167018
                5120828
                27880800
                d9ce5e77-2deb-403a-9830-d8be9a23c114
                © 2016 Miller et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 August 2016
                : 7 November 2016
                Page count
                Figures: 6, Tables: 2, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 092872/Z/10/Z
                Award Recipient :
                This work has been supported by Oxford Glycobiology Institute Endowment and a research grant from Emergent BioSolutions, Inc. (formerly Unither Virology LLC). ACS was funded by a Clarendon Fund Scholarship and a Santander Graduate Scholarship from Pembroke College, Oxford. SGS was funded by the Wellcome Trust, grant 092872/Z/10/Z. NZ is a Fellow of Merton College, Oxford. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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