title: Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.
abstract: Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.
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.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Wentao
authors.initials: W
authors.lastname: Li
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Lione
authors.initials: L
authors.lastname: Willems
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Tom J
authors.initials: TJ
authors.lastname: Meuleman
authors.affiliation: Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Wubbolts
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Frank J M
authors.initials: FJ
authors.lastname: van Kuppeveld
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands b.j.bosch@uu.nl.
authors.forename: Berend Jan
authors.initials: BJ
authors.lastname: Bosch
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D053485:Porcine epidemic diarrhea virus / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D064370:Spike Glycoprotein, Coronavirus / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D014709:Vero Cells; D014776:Virus Cultivation / Q000379:methods; D053586:Virus Internalization
chemical_list: D064370:Spike Glycoprotein, Coronavirus; D014357:Trypsin