Aminopeptidase N is not required for porcine epidemic diarrhea virus cell entry

Abstract

Porcine epidemic diarrhea virus (PEDV) is an emerging pathogenic coronavirus that causes a significant economic burden to the swine industry. The virus infects the intestinal epithelium and causes villous atrophy, resulting in diarrhea and dehydration. Interaction of the viral spike (S) surface glycoprotein - through its S1 subunit - with the host cell receptor is the first step in infection and the main determinant for virus tropism. As for several other alphacoronaviruses including the porcine transmissible gastroenteritis virus (TGEV) and the human coronavirus 229E (HCoV-229E), the aminopeptidase N (APN) protein was reported to be a functional receptor for PEDV. In this study we examined the role of APN as a receptor. We show that overexpression of porcine APN renders MDCK cells susceptible to TGEV, but not to PEDV. Consistently, unlike TGEV-S1, PEDV-S1 exhibited no binding to cell-surface expressed APN or to a soluble version of APN. Moreover, preincubation of these viruses with soluble APN or pretreatment of APN expressing ST cells with soluble TGEV-S1 blocked TGEV infection, but had no effect on infection by PEDV. The combined observations indicated that APN is not required for PEDV infection. To definitively prove this conclusion, we applied CRISPR/Cas9 genome engineering to knock out APN expression in PEDV-susceptible porcine (ST) and human cell lines (Huh7 and HeLa). As a consequence these cells no longer bound TGEV-S1 and HCoV-229E-S1 at their surface and were resistant to infection by the corresponding viruses. However, genetic ablation of APN expression had no effect on their infectability by PEDV, demonstrating that APN is not essential for PEDV cell entry.

Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility

“…PDCoV can replicate in swine testis (ST) cells with supplemental trypsin (23). To determine the role of pAPN interaction in PDCoV entry, we used a mutant ST cell line lacking cell surface APN expression (ST-pAPN KO ) that had been made previously using CRISPR/ Cas9 genome editing (44). In addition, we generated an ST-pAPN KO cell line with reconstituted pAPN expression (ST-pAPN KO -pAPN).…”

“…In addition, we generated an ST-pAPN KO cell line with reconstituted pAPN expression (ST-pAPN KO -pAPN). Integrity of the mutant cell lines was confirmed by sequencing, TGEV S1 cell surface staining (44)…”

The enhanced replication of an S-intact PEDV during coinfection with an S1 NTD-del PEDV in piglets

“…S1 subunit contains two domains, the N-terminal domain (S1 NTD, residues 21-324 based on PEDV CV777) and the C-terminal domain (S1 CTD, residues 253-638). However, the cellular receptor of PEDV is still unknown (Li et al, 2017b;Shirato et al, 2016). Although the sialic acid binding activity of S1-NTD was observed in many coronaviuses and facilitated virus replication (Schwegmann-Wessels et al, 2011;Li et al, 2016;Desmarets et al, 2014), S1-NTD is dispensable for some mutants of transmissible gastroenteritis coronavirus (TGEV) and PEDV (Schwegmann-Wessels et al, 2011;Oka et al, 2014;Suzuki et al, 2016;Hou et al, 2017).…”

Characterization of the interaction between recombinant porcine aminopeptidase N and spike glycoprotein of porcine epidemic diarrhea virus

“…[32,41], are efficiently infected by PEDV, and widely utilized for isolation and series propagation of the virus [41,[65][66][67][68]. Recently, no effects were reported on the susceptibility to PEDV after knocking out APN in porcine swine testis cells and human cell lines (human hepatoma cells, Huh7 cells and human cervical cancer cells, HeLa cells) by CRISPR/Cas9 genome editing [38]. Moreover, soluble pAPN ectodomain neither interacted with PEDV nor inhibited its infection [37].…”

“…PEDV S1-CTD (residues 505-629) was further demonstrated to interact with pAPN ectodomain (residues 63-963) [36]. Interestingly, a few recent studies show that pAPN is not the functional receptor for PEDV infection [37,38]. However, there is a lack of direct demonstration of the interaction between pAPN and PEDV S protein to clarify this discrepancy.…”