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      Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis

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          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 growth in vitro and reduced weight loss and clinical signs in an in vivo murine 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.

          IMPORTANCE Cellular retrograde transport pathways traffic cargo from endosomes to the trans-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.

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          Most cited references 42

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          The formation and function of extracellular enveloped vaccinia virus.

          Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.
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            Tracing the retrograde route in protein trafficking.

            Retrograde transport, in which proteins and lipids are shuttled between endosomes and biosynthetic/secretory compartments such as the Golgi apparatus, is crucial for a diverse range of cellular functions. Mechanistic studies that explore the molecular machinery involved in this retrograde trafficking route are shedding light on the functions of transport proteins and are providing fresh insights into possible new therapeutic directions.
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              Inhibition of retrograde transport protects mice from lethal ricin challenge.

              Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target. Copyright 2010 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                J Virol
                J. Virol
                jvi
                jvi
                JVI
                Journal of Virology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0022-538X
                1098-5514
                31 August 2016
                28 October 2016
                15 November 2016
                28 October 2016
                : 90
                : 22
                : 10120-10132
                Affiliations
                [a ]The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom
                [b ]The Pirbright Institute, Pirbright, Surrey, United Kingdom
                [c ]SCBM, Institute of Biology and Technology of Saclay, CEA, LabEx LERMIT, Université Paris-Saclay, Gif Sur Yvette, France
                [d ]SIMOPRO, Institute of Biology and Technology of Saclay, CEA, LabEx LERMIT, Université Paris-Saclay, Gif Sur Yvette, France
                [e ]Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
                University of Florida
                Author notes
                Address correspondence to Philippa M. Beard, pip.beard@ 123456roslin.ed.ac.uk .

                Citation Harrison K, Haga IR, Pechenick Jowers T, Jasim S, Cintrat J-C, Gillet D, Schmitt-John T, Digard P, Beard PM. 2016. Vaccinia virus uses retromer-independent cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions during virus morphogenesis. J Virol 90:10120–10132. doi: 10.1128/JVI.01464-16.

                Article
                01464-16
                10.1128/JVI.01464-16
                5105650
                27581988
                Copyright © 2016 Harrison et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 49, Pages: 13, Words: 10144
                Product
                Funding
                This work was supported by strategic program grants BBS/E/D/20241864 and BBS/E/D/20241866 from the BBSRC to P.M.B. and P.D. and a University of Edinburgh Principal's Career Development Ph.D. Scholarship to K.H. Financial support was also provided by the Joint Ministerial Program of R&D against CBRNE risks, ANR grant Anti-HUS ANR-14-CE16-0004, Lermit LabEx grant R3 RetroLeishma, Ile de France Region grant from the DIM Malinf Initiative 140101, and CEA.
                Categories
                Virus-Cell Interactions
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                Microbiology & Virology

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