title: Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis.
abstract: Poxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growthin vitroand reduced weight loss and clinical signs in anin vivomurine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.IMPORTANCECellular retrograde transport pathways traffic cargo from endosomes to thetrans-Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small-molecule inhibitors reduced the replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the wrapping step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of antipoxvirus drugs.
authors: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.
authors: Kate
authors: K
authors: Harrison
authors: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.|The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors: Ismar R
authors: IR
authors: Haga
authors: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.
authors: Tali
authors: T
authors: Pechenick Jowers
authors: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.
authors: Seema
authors: S
authors: Jasim
authors: SCBM, Institute of Biology and Technology of Saclay, CEA, LabEx LERMIT, Université Paris-Saclay, Gif Sur Yvette, France.
authors: Jean-Christophe
authors: JC
authors: Cintrat
authors: SIMOPRO, Institute of Biology and Technology of Saclay, CEA, LabEx LERMIT, Université Paris-Saclay, Gif Sur Yvette, France.
authors: Daniel
authors: D
authors: Gillet
authors: Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors: Thomas
authors: T
authors: Schmitt-John
authors: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.
authors: Paul
authors: P
authors: Digard
authors: The Pirbright Institute, Pirbright, Surrey, United Kingdom pip.beard@roslin.ed.ac.uk.|The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom.
authors: Philippa M
authors: PM
authors: Beard