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. IMPORTANCERecurring 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. P orcine epidemic diarrhea virus (PEDV) belongs to the genusAlphacoronavirus in the family Coronaviridae and is the causative agent of porcine epidemic diarrhea (1). The virus is prevalent in East Asia, inflicting severe economic damage due to high mortality rates in young piglets, and recently made its first appearance on the North American subcontinent (2-4). PEDV infects the epithelia of the small intestine, an environment rich in proteases, and causes villous atrophy, resulting in diarrhea and dehydration. Intriguingly, in vitro propagation of PEDV isolates requires supplementation of trypsin to the cell culture supernatant (5). It has been hypothesized that trypsin mediates activation of virions for membrane fusion by cleaving the spike (S) glycoprotein (5, 6). Trimeric S proteins decorate the virion envelope and mediate receptor binding and membrane fusion. The S protein has been recognized as a class I fusion protein by its molecular features (7,8).Class I fusion proteins are generated in a locked conformation to prevent premature triggering of the fusion mechanism and are subsequently prepared for action by proteolytic processing, a step called priming (reviewed in reference 9). This cleavage is separating two functionally distinct protein domains, a soluble head domain responsible for receptor binding and a membrane bound subunit comprising the fusion machinery. A characteristic feature of the cleaved, fusion-ready subunit is an N-terminal fusion peptide. Proteolytic priming can occur in the virus-producing cell, in the extracellular environment, or after contact with the target cell membrane. Priming of the PEDV S protein is ...

Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage

“…The S protein on the viral surface has the highest sequence diversity among PEDV structural proteins. It is a glycoprotein that binds to the host cell receptors and induces the subsequent virus-cell membrane fusion (Wicht et al, 2014). Several neutralizing epitopes, including the CO-26 K equivalent epitope (COE) (aa position 499-638), SS2 (aa 748-755), SS6 (aa 746-771), and 2C10 (aa 1368-1374) have been identified (Oh et al, 2014;Sun et al, 2007).…”

Porcine epidemic diarrhoea virus with a recombinant S gene detected in Hungary, 2016

“…In addition, there is another ORF named ORF3 between the S and E genes, encoding an ion channel, which possibly regulates virus production (Wang et al, 2012). Among these the main research interest is focused on the S gene and its glycoprotein product, which in the presence of proteolytic trypsin is cleaved into two functionally distinct subunits, S1 and S2, responsible for receptor binding and fusion mechanisms, respectively, which makes the spike protein the primary target for neutralising antibodies (Wicht et al, 2014).…”

Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses

“…It can be divided into S1 and S2 domains. In some coronaviruses the S protein is processed into S1 and S2 fragments by cellular proteases or trypsin (Belouzard et al, 2012;Wicht et al, 2014). The S protein is a major target for virus neutralizing antibodies (Chang et al, 2002;Reguera et al, 2012).…”