Biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion
The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular targ...
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| creator | Thomas, Celestine J Shankar, Sundaresh Casquilho-Gray, Hedi E York, Joanne Sprang, Stephen R Nunberg, Jack H |
| description | The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein. |
| doi_str_mv | 10.1371/journal.pone.0051114 |
| format | Article |
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The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Thomas, Celestine J</au><au>Shankar, Sundaresh</au><au>Casquilho-Gray, Hedi E</au><au>York, Joanne</au><au>Sprang, Stephen R</au><au>Nunberg, Jack H</au><au>Liu, Shan-Lu</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-11-30</date><risdate>2012</risdate><volume>7</volume><issue>11</issue><spage>e51114</spage><epage>e51114</epage><pages>e51114-e51114</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23226473</pmid><doi>10.1371/journal.pone.0051114</doi><tpages>e51114</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Antibodies, Neutralizing - immunology Antiviral agents Biochemistry Biology Biotechnology Cell fusion Chlorocebus aethiops Fever Furin - metabolism Glycoproteins Glycoproteins - metabolism Hemorrhage Hemorrhagic fever Hemorrhagic fevers HIV Human immunodeficiency virus Humans Hydrogen-Ion Concentration Immunoglobulins Influenza Inhibition Junin virus - metabolism Lipids - chemistry Medicine Membrane Fusion Monoclonal antibodies Mutant Proteins - metabolism Neutralization Tests Peptides pH effects Protein Conformation Proteins Proteolipids - metabolism Proteolysis Public health Recombinant Proteins - metabolism Structural analysis Surface Plasmon Resonance Vero Cells Viral Envelope Proteins - metabolism Virions Virus Shedding Viruses |
| title | Biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion |
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