Mechanistic Insight Into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc
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Mathematisch-Naturwissenschaftliche Fakultät Pablo Guardado-Calvo | Eduardo A. Bignon | Eva Stettner Scott Allen Jeffers | Jimena PeÂrez-Vargas | Gerard Pehau-Arnaudet M. Alejandra Tortorici | Jean-Luc Jestin | Patrick England Nicole D. Tischler | Félix A. Rey Mechanistic insight into bunyavirus- induced membrane fusion from structure-function analyses of the hantavirus envelope glycoprotein Gc Suggested citation referring to the original publication: PLoS Pathogens 12 (2016) 10, Art. e1005813 DOI https://doi.org/10.1371/journal.ppat.1005813 ISSN 1553-7374 Postprint archived at the Institutional Repository of the Potsdam University in: Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe ; 676 ISSN 1866-8372 https://nbn-resolving.org/urn:nbn:de:kobv:517-opus4-411599 DOI https://doi.org/10.25932/publishup-41159 RESEARCH ARTICLE Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc Pablo Guardado-Calvo1,2☯, Eduardo A. Bignon3☯, Eva Stettner1¤a, Scott Allen Jeffers1¤b, Jimena PeÂrez-Vargas1,2¤c, Gerard Pehau-Arnaudet4,5, M. Alejandra Tortorici1,2, Jean- Luc Jestin1¤d, Patrick England5,6, Nicole D. Tischler3*, FeÂlix A. Rey1,2* a11111 1 Institut Pasteur, Unite de Virologie Structurale, DeÂpartement de Virologie, Paris, France, 2 CNRS UMR3569 Virologie, Paris, France, 3 FundacioÂn Ciencia & Vida, Molecular Virology Laboratory, Santiago, Chile, 4 Institut Pasteur, Ultrapole, DeÂpartement de Biologie Cellulaire et Infection, Paris, France, 5 CNRS UMR 3528, Paris, France, 6 Institut Pasteur, Plateforme de Biophysique des MacromoleÂcules et de leurs Interactions, DeÂpartement de Biologie Structurale et Chimie, Paris, France ☯ These authors contributed equally to this work. ¤a Current address: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany OPEN ACCESS ¤b Current address: Brammer Bio LLC, Alachua, Florida, United States of America Citation: Guardado-Calvo P, Bignon EA, Stettner E, ¤c Current address: CIRI, International Centre for Infectiology Research, team EVIR, Inserm U1111, Jeffers SA, PeÂrez-Vargas J, Pehau-Arnaudet G, et Universite de Lyon, Lyon, France ¤d Current address: Institut des Hautes Etudes Scientifiques, Bures-sur-Yvette, France al. (2016) Mechanistic Insight into Bunyavirus- * [email protected] (NDT); [email protected] (FAR) Induced Membrane Fusion from Structure- Function Analyses of the Hantavirus Envelope Glycoprotein Gc. PLoS Pathog 12(10): e1005813. doi:10.1371/journal.ppat.1005813 Abstract Editor: Richard J. Kuhn, Purdue University, UNITED STATES Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of Received: June 14, 2016 hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with Accepted: July 17, 2016 high case fatality rates. There is only limited knowledge about the organization of the viral Published: October 26, 2016 particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the func- Copyright: © 2016 Guardado-Calvo et al. This is an tion of which is essential for virus entry. We describe here the X-ray structures of Gc from open access article distributed under the terms of Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral the Creative Commons Attribution License, which pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The permits unrestricted use, distribution, and reproduction in any medium, provided the original structures confirm the prediction that Gc is a class II fusion protein, containing the charac- author and source are credited. teristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of Data Availability Statement: All relevant data are arboviruses such as alpha- and flaviviruses. The structures also show a number of features within the paper and its Supporting Information of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts files. The coordinate files of the structures residues from three different loops into the target membrane to drive fusion, as confirmed described in this manuscript have been submitted functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of to the Protein Data Bank, and the corresponding accession codes are listed in the S1 Table. having a single ªfusion loopº. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Fur- Funding: FAR and PGC acknowledge support the Infect-ERA IMI European network, program thermore, the structure reveals that hantavirus Gc has an additional N-terminal ªtailº that is ªHantaHuntº and its coordinator, Professor crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the Andreas Herrmann. FAR also received funding quaternary arrangement of the trimer as compared to the standard class II fusion proteins. from the "Integrative Biology of Emerging PLOS Pathogens | DOI:10.1371/journal.ppat.1005813 October 26, 2016 1 / 32 X-Ray Structures and Functional Dissection of the Hantavirus Fusion Protein Infectious Diseases" Labex (Laboratoire The mechanistic understandings derived from these data are likely to provide a unique han- d'Excellence) grant NÊ ANR-10-LABX-62-IBEID dle for devising treatments against these human pathogens. (French Government's ªInvestissements d'Avenirº program). ES was funded as ªExperienced Researcherº (ER) by the Marie Curie Training Network ªVirus Entryº (Call:FP7-PEOPLE-2007-1- 1-ITN. NDT received support from FONDECYT Author Summary 1140050 and Basal PFB-16 grants from CONICYT (Chile). The funders had no role in study design, Hantaviruses belong to the Bunyaviridae family of enveloped viruses. This family englobes data collection and analysis, decision to publish, or in total five established genera: Tospovirus (infecting plants), and Phlebovirus, Orthobu- preparation of the manuscript. nyavirus, Nairovirus and Hantavirus infecting animals, some of which cause serious dis- Competing Interests: I have read the journal's ease in humans. An important characteristic of the hantaviruses is that they are not policy and I understand that one of the authors of transmitted to humans by arthropod vectors, as those from the other genera, but by direct this manuscript (SAJ) has competing interests exposure to excretions from infected small mammals. As all enveloped viruses, they because of being employed by Brammer Bio LLC, 13702 Innovation Drive Alachua FL 32615. require the activity of a membrane fusogenic protein, Gc, for entry into their target cells. Our structural analysis of the hantavirus fusion protein Gc led to the identification of a conserved pattern of cysteines involved in disulfide bonds stabilizing the Gc fold. This motif is matched exclusively by all of the available bunyavirus Gc sequences in the data- base, with the notable exception of phlebovirus Gc, which appears closer in structure to the fusion proteins of other families of arthropod-borne viruses, such as the flaviviruses and alphaviruses. This analysis further suggests mechanistic similarities with hantaviruses in the fusion mechanism of viruses in the remaining three most closely related bunyavirus genera, which we propose belong to a new separate sub-class of fusion proteins with a mul- tipartite membrane targeting region. Introduction Hantaviruses are a small group of zoonotic viruses of rodents, bats and insectivores such as moles and shrews [1]. They are often transmitted to humans by persistently infected rodents, causing serious outbreaks of pulmonary syndrome or of hemorrhagic disease with renal syn- drome [2, 3]. The case fatality rates can reach 50%, for instance in the case of the “Sin Nombre” hantavirus outbreak in the 1990s in the four-corners area in the US [4]. The name hantavirus derives from the prototype virus, Hantaan virus, which was discovered in the early 1950s dur- ing the Korean war, when troops stationed by the Hantaan river developed hemorrhagic mani- festations [5]. Outbreaks of hantavirus disease of varying severity have occurred periodically in the last decades throughout the Americas [6, 7] and in Europe and Asia [8, 9]. It is therefore important to understand the structural organization of hantavirus particles as a step forward in attempts to devise curative or preventative strategies. Hantaviruses constitute one of five genera forming the Bunyaviridae family of enveloped RNA viruses, which have a genome composed of three segments of single-stranded RNA of negative polarity [10]. The bunyavirus proteins involved in genome replication–the large (L) polymerase and the nucleocapsid (N) protein, encoded in the large and small genome segment, respectively–are very similar to their counterparts in other families of segmented negative- strand (sns)RNA viruses, such as the Arenaviridae or the Orthomyxoviruses [11]. In contrast, the envelope glycoproteins, which derive from a polyprotein precursor encoded in the medium (M) size genomic segment, are totally unrelated. Whereas the other snsRNA virus families dis- play class I membrane fusion proteins characterized by a central alpha-helical coiled-coil in their post-fusion form, the bunyavirus envelope proteins have properties of class II enveloped