Journal of Virology 2014-07-01

Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

Oliver Wicht, Wentao Li, Lione Willems, Tom J Meuleman, Richard W Wubbolts, Frank J M van Kuppeveld, Peter J M Rottier, Berend Jan Bosch

Index: J. Virol. 88(14) , 7952-61, (2014)

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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.Copyright © 2014, American Society for Microbiology. All Rights Reserved.