Receptor usage and cell entry of porcine epidemic diarrhea coronavirus

Abstract

Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread.

FIG 1

PEDV spike protein. (A) Domain structure of PEDV spike. It contains a receptor-binding S1 subunit, a membrane fusion S2 subunit, a single-pass transmembrane anchor (TM), and a short intracellular tail (IC). S1 contains an N-terminal domain (S1-NTD) and a C-terminal domain (S1-CTD). S2 contains the fusion peptide (FP), heptad repeat 1 (HR1), and heptad repeat 2 (HR2), all of which are essential structural elements for the membrane fusion process. (B) Amino acid sequence identities between PEDV spike and the spikes from TGEV, BtCoV/512/2005, and HCoV-NL63 in different regions. GenBank accession numbers are AGO58924.1 for PEDV spike, CAA29175.1 for TGEV spike, ABG47078.1 for BtCoV/512/2005 spike, and AAS58177.1 for HCoV-NL63 spike.

FIG 2

PEDV spike binds porcine APN, human APN, and sugar receptors. (A) SDS-PAGE analysis of recombinant PEDV S1-NTD-CTD and TGEV S1-NTD-CTD. Both proteins were fused to a C-terminal human IgG1 Fc tag. The gel was stained using Coomassie blue. Numbers at the left are molecular masses (in kilodaltons). (B) Dot blot hybridization assay showing the interactions between PEDV or TGEV S1-NTD-CTD (with a C-terminal human IgG1 Fc tag) and porcine APN (pAPN) or human APN (hAPN) (with a C-terminal His₆ tag) using a procedure as previously described (24). APN-binding S1-NTD-CTDs were detected using antibodies against their C-terminal Fc tag and subsequently subjected to enzymatic color reactions. Bovine serum albumin (BSA) was used as a negative control. (C) Dot blot hybridization assay showing the interactions between PEDV or TGEV S1-NTD-CTD and sugar moieties on mucin-spotted nitrocellulose membranes using a procedure as previously described (25). Mucin was either mock treated or treated with neuraminidase (New England BioLabs Inc.). Sugar-binding S1-NTD-CTDs were detected using antibodies against their C-terminal Fc tag and subsequently subjected to enzymatic color reactions. (D) A glycan screen array was performed to identify the type(s) of sugar most favored by PEDV S1-NTD-CTD (with a C-terminal Fc tag) using a procedure as previously described (26). A glycan library composed of 609 different natural and synthetic mammalian glycans (see Table S1 in the supplemental material) was screened for PEDV S1-NTD-CTD binding. Glycan-binding S1-NTD-CTD was detected using antibodies against its C-terminal Fc tag. The readout was described arbitrarily as relative fluorescence units (RFU). Among these glycans, N-acetylneuraminic acid (Neu5Ac) shows the highest binding affinity for PEDV S1-NTD-CTD.

FIG 3

PEDV spike-mediated pseudovirus entry into host cells. PEDV spike- and TGEV spike-pseudotyped retroviruses were produced and used to infect cells using a procedure as previously described (27). Trypsin was not included in the pseudovirus entry assay. The cells being infected were MDCK cells exogenously expressing human APN (hAPN), porcine APN (pAPN), or an empty vector (A), PK-15 cells (B), and Huh-7 cells (C). For antibody inhibition, cells were preincubated with 20 μg/ml anti-human APN antibody (Santa Cruz Biotechnology) for 1 h at 37°C before pseudovirus infection. For mucin inhibition, PEDV spike- or TGEV spike-pseudotyped retroviruses were preincubated with 500 μg/ml porcine or bovine mucin before they were used to infect cells. The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry. Error bars indicate standard errors of the means (SEM) (n = 4).

FIG 4

PEDV infections in cell culture. PEDV strain Ohio VBS2 was used to infect different cell lines at an MOI of 1.0 using a procedure as previously described (30). Trypsin (5 μg/ml) was included in the cell culture medium to facilitate live-PEDV infections. Twenty-four hours postinoculation, cells were fixed with 4.0% (vol/vol) paraformaldehyde and 0.2% (vol/vol) glutaraldehyde. PEDV was detected with fluorescein isothiocyanate (FITC)-labeled mouse anti-PEDV N protein antibody and observed under a fluorescence microscope.