title: Identification and characterization of a proteolytically primed form of the murine coronavirus spike proteins after fusion with the target cell.
abstract: Enveloped viruses carry highly specialized glycoproteins that catalyze membrane fusion under strict spatial and temporal control. To prevent premature activation after biosynthesis, viral class I fusion proteins adopt a locked conformation and require proteolytic cleavage to render them fusion-ready. This priming step may occur during virus exit from the infected cell, in the extracellular milieu or during entry at or in the next target cell. Proteolytic processing of coronavirus spike (S) fusion proteins during virus entry has been suggested but not yet formally demonstrated, while the nature and functionality of the resulting subunit is still unclear. We used a prototype coronavirus--mouse hepatitis virus (MHV)--to develop a conditional biotinylation assay that enables the specific identification and biochemical characterization of viral S proteins on virions that mediated membrane fusion with the target cell. We demonstrate that MHV S proteins are indeed cleaved upon virus endocytosis, and we identify a novel processing product S2* with characteristics of a fusion-active subunit. The precise cleavage site and the enzymes involved remain to be elucidated.
  
  
  OBJECTIVE
  Virus entry determines the tropism and is a crucial step in the virus life cycle. We developed an approach to characterize structural components of virus particles after entering new target cells. A prototype coronavirus was used to illustrate how the virus fusion machinery can be controlled.
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Oliver
authors.initials: O
authors.lastname: Wicht
authors.forename: Christine
authors.initials: C
authors.lastname: Burkard
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.forename: Frank J M
authors.initials: FJ
authors.lastname: van Kuppeveld
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
authors.forename: Berend Jan
authors.initials: BJ
authors.lastname: Bosch
mesh_terms: D000818:Animals; D004705:Endocytosis; D051379:Mice; D006517:Murine hepatitis virus / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D064370:Spike Glycoprotein, Coronavirus / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D064370:Spike Glycoprotein, Coronavirus