title: Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state.
abstract: Vesicular stomatitis virus (VSV; the prototype rhabdovirus) fusion is triggered at low pH and mediated by glycoprotein G, which undergoes a low-pH-induced structural transition. A unique feature of rhabdovirus G is that its conformational change is reversible. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. The crystal structures of G pre- and postfusion states have been elucidated, leading to the identification of several acidic amino acid residues, clustered in the postfusion trimer, as potential pH-sensitive switches controlling the transition back toward the prefusion state. We mutated these residues and produced a panel of single and double mutants whose fusion properties, conformational change characteristics, and ability to pseudotype a virus lacking the glycoprotein gene were assayed. Some of these mutations were also introduced in the genome of recombinant viruses which were further characterized. We show that D268, located in the segment consisting of residues 264 to 273, which refolds into postfusion helix F during G structural transition, is the major pH sensor while D274, D395, and D393 have additional contributions. Furthermore, a single passage of recombinant virus bearing the mutation D268L (which was demonstrated to stabilize the G postfusion state) resulted in a pseudorevertant with a compensatory second mutation, L271P. This revealed that the propensity of the segment of residues 264 to 273 to refold into helix F has to be finely tuned since either an increase (mutation D268L alone) or a decrease (mutation L271P alone) of this propensity is detrimental to the virus.
  
  
  OBJECTIVE
  Vesicular stomatitis virus enters cells via endocytosis. Endosome acidification induces a structural transition of its unique glycoprotein (G), which mediates fusion between viral and endosomal membranes. G conformational change is reversible upon increases in pH. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. We mutated five acidic residues, proposed to be pH-sensitive switches controlling the structural transition back toward the prefusion state. Our results indicate that residue D268 is the major pH sensor, while other acidic residues have additional contributions, and reveal that the propensity of the segment consisting of residues 264 to 273 to adopt a helical conformation is finely regulated. This segment might be a good target for antiviral compounds.
authors.affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France.
authors.forename: Anna
authors.initials: A
authors.lastname: Ferlin
authors.affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Raux
authors.affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France.
authors.forename: Eduard
authors.initials: E
authors.lastname: Baquero
authors.affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France.
authors.forename: Jean
authors.initials: J
authors.lastname: Lepault
authors.affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France gaudin@vms.cnrs-gif.fr.
authors.forename: Yves
authors.initials: Y
authors.lastname: Gaudin
mesh_terms: D000818:Animals; D002460:Cell Line; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation; D018116:Vesiculovirus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D053586:Virus Internalization
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D014759:Viral Envelope Proteins