Identification of the functional domain of the porcine epidemic diarrhoea virus receptor

Abstract

Porcine aminopeptidase N (pAPN) is a functional receptor for porcine epidemic diarrhoea virus (PEDV). Although PEDV is known to use the pAPN as the major receptor for cell entry, the crucial domain of the pAPN that interacts with the PEDV is still unknown. In the present study, in order to determine the crucial domain of the pAPN, the extracellular domain of pAPN was divided into three subdomains named SPA, SPB and SPC, based on its secondary structure. Recombinant plasmid pcDNA3.1 expressing SPA, SPB and SPC was constructed and introduced into Madin-Darby canine kidney (MDCK) cells by transfection. Following the detection of PEDV infection in transfected MDCK cells after PEDV challenge, we clearly demonstrated that the SPC subdomain plays a key role in cell entry of PEDV and its expression permits PEDV growth in transfected MDCK cells, while virus propagation can be inhibited by anti-SPC serum, indicating that the SPC subdomain appears to be a crucial functional domain in contributing to efficient PEDV infection. Porcine epidemic diarrhoea virus (PEDV) belonging to the group I coronavirus is the major cause of lethal diarrhoea disease in piglets (Oh et al., 2003;Weiss & Navas-Martin, 2005). Like transmissible gastroenteritis virus, PEDV uses porcine aminopeptidase N (pAPN) as the major receptor for cell entry (Chen et al., 2012;Delmas et al., 1992Delmas et al., , 1993Hansen et al., 1998;Li et al., 2007; Reguera et al., 2012;Schultze et al., 1995;van der Hoek et al., 2004). Remarkably, it was found that the two viruses utilized distinct cultured cell lines for virus propagation in vitro (Delmas et al., 1992;Hofmann & Wyler, 1988). pAPN, characterized as a member of a membrane-bound metalloprotease family, is expressed predominantly on the epithelial cell surface of the kidney, small intestine and respiratory tract (Kenny & Maroux, 1982;Lendeckel et al., 2000;Nam & Lee, 2010). Although pAPN is recognized as the major cell receptor for PEDV entry and initiates cell infection through interaction between the spike protein of PEDV and pAPN (Bosch et al., 2003;Godet et al., 1994), the crucial domain of pAPN that interacts with PEDV is still unknown.In this study, in order to determine the crucial domain of pAPN that interacts with PEDV, we obtained the protein structure set of pAPN from the SWISS-MODEL web server as described previously (Arnold et al., 2006;Benkert et al., 2011;Biasini et al., 2014), and its transmembrane domains, secondary structure and functional domains were analysed using TMHMM 2.0, the PSIpred-MEMSAT 3 web server and the Predict Protein server, respectively. As shown in Fig. 1(a), the pAPN encompassed an intracellular domain comprising residues 1-11 and an extracellular domain, in which the residues 12-35 forming an a-helix were considered as the transmembrane domain of pAPN. The extracellular domain of pAPN was divided into three subdomains named SPA, SPB and SPC, respectively. SPA (residues 72-369) contained a 15-stranded bbarrel fold, showing structural evidence for the common ancestry of M1 ...

Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets

“…Such diverse tissue tropisms may be due to different receptor usage and/or proteolytic processing of the S proteins between PEDV and TGEV. For many years, the TGEV receptor, porcine aminopeptidase N (pAPN), was also considered to be the putative receptor of PEDV (40)(41)(42)(43). However, recent studies revealed that PEDV could infect pAPN knockout cell lines (11), but the protease activity of pAPN enhanced PEDV infection (10), suggesting that pAPN is not the cellular receptor for PEDV.…”

Porcine amino peptidase N domain VII has critical role in binding and entry of porcine epidemic diarrhea virus

“…They also reported the C-terminal ␣domain might be important as the active site. More recently, Shan et al reported the protein structure on pAPN by SWISS-MODEL web server (Shan et al, 2015). They reported pAPN contains small intra-cellular domain and large extra cellular domain divided into 3 subdomains named SPA, SPB and SPC.…”

“…Indeed, even new isolates of PEDV are able to show distinct signs of CPE in Vero cells only after several passages (Pan et al, 2010), which could probably be an adaptation period. As the functional domain study on pAPN, Shan et al reported that SPC region on pAPN might be putative functional domain for PEDV infection (Shan et al, 2015). They found MDCK cells expressing only SPC region successfully infected by PEDV and even shown cytopathic effects (CPE).…”

Construction and characterization of thymidine auxotrophic (ΔthyA) recombinant Lactobacillus casei expressing bovine lactoferricin

“…Indirect immunofluorescence assay was performed to detect the expression of Lfcin on the cell surface of ΔthyA L.casei -LFEC as previously described [ 19 ]. Briefly, 1 mL of ΔthyA L.casei -LFEC cultured in GM17 broth for 12 h was centrifuged, washed with PBS three times, and resuspended in 1 mL of sterile PBS-3% bovine serum albumin (BSA) containing mouse anti-myc antibody; following the incubation at 37°C for 1 h, the cells were harvested, washed three times, and incubated in 1 mL of FITC-conjugated goat anti-mouse IgG antibody (diluted at 1:500) at 37°C for 1 h; then, the cells were washed three times, transferred onto a glass slide, and fixed with cold acetone for 30 min; Confocal microscope was used to observe fluorescence signals.…”