title: ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.
abstract: In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.
  
  
  OBJECTIVE
  Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.
authors: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Christine
authors: C
authors: Burkard
authors: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Monique H
authors: MH
authors: Verheije
authors: Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands.
authors: Bart L
authors: BL
authors: Haagmans
authors: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Frank J
authors: FJ
authors: van Kuppeveld
authors: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Peter J M
authors: PJ
authors: Rottier
authors: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Berend-Jan
authors: BJ
authors: Bosch
authors: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands c.a.m.dehaan@uu.nl.
authors: Cornelis A M
authors: CA
authors: de Haan
mesh_terms: D000818:Animals; D002018:Bufanolides / Q000494:pharmacology; D002301:Cardiac Glycosides / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003333:Coronaviridae Infections / Q000503:physiopathology*; D016765:Coronavirus, Feline / Q000502:physiology; D020868:Gene Silencing; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D006517:Murine hepatitis virus / Q000502:physiology; D010042:Ouabain / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014709:Vero Cells; D053585:Virus Attachment / Q000187:drug effects*; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: D002018:Bufanolides; D002301:Cardiac Glycosides; D034741:RNA, Small Interfering; D010042:Ouabain; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase; C022777:bufalin
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