title: 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.
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Wentao
authors.initials: W
authors.lastname: Li
authors.affiliation: State Key laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei Province, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Luo
authors.affiliation: State Key laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei Province, China.
authors.forename: Qigai
authors.initials: Q
authors.lastname: He
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Frank J M
authors.initials: FJM
authors.lastname: van Kuppeveld
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Peter J M
authors.initials: PJM
authors.lastname: Rottier
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Electronic address: b.j.bosch@uu.nl.
authors.forename: Berend-Jan
authors.initials: BJ
authors.lastname: Bosch
mesh_terms: D000818:Animals; D018826:CD13 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D015870:Gene Expression; D055786:Gene Knockout Techniques; D006801:Humans; D053485:Porcine epidemic diarrhea virus / Q000502:physiology*; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D053586:Virus Internalization
chemical_list: D011991:Receptors, Virus; D018826:CD13 Antigens
keywords: Aminopeptidase N; Coronavirus; Porcine epidemic diarrhea virus; Receptor; Virus cell entry