title: Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor.
abstract: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens.
  
  
  OBJECTIVE
  The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Yi-Ying
authors.initials: YY
authors.lastname: Chou
authors.affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Cuevas
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Margot
authors.initials: M
authors.lastname: Carocci
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Stubbs
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Minghe
authors.initials: M
authors.lastname: Ma
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: David K
authors.initials: DK
authors.lastname: Cureton
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Luke
authors.identifier: http://orcid.org/0000-0002-4849-4148
authors.initials: L
authors.lastname: Chao
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Frances
authors.initials: F
authors.lastname: Evesson
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Kangmin
authors.initials: K
authors.lastname: He
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Priscilla L
authors.initials: PL
authors.lastname: Yang
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Whelan
authors.affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Susan R
authors.identifier: http://orcid.org/0000-0002-3094-3769
authors.initials: SR
authors.lastname: Ross
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA kirchhausen@crystal.harvard.edu rgaudin@unistra.fr.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.|Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Kirchhausen
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA kirchhausen@crystal.harvard.edu rgaudin@unistra.fr.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Raphaël
authors.initials: R
authors.lastname: Gaudin
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000821:virology; D055786:Gene Knockout Techniques; D006478:Hemorrhagic Fever, American / Q000235:genetics / Q000378:metabolism / Q000821:virology; D006801:Humans; D018051:Junin virus / Q000187:drug effects / Q000502:physiology; D051379:Mice; D011485:Protein Binding; D021381:Protein Transport; D059748:Proteolysis; D012261:Ribonucleoproteins / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D019562:Viral Load; D014764:Viral Proteins / Q000378:metabolism; D053585:Virus Attachment / Q000187:drug effects; D053586:Virus Internalization / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D000096926:Benzenesulfonamides
chemical_list: D000199:Actins; D000998:Antiviral Agents; D001549:Benzamides; D002966:Clathrin; D012261:Ribonucleoproteins; D013890:Thiourea; D014764:Viral Proteins; D020764:cdc42 GTP-Binding Protein; C000610025:ZCL278; D000096926:Benzenesulfonamides