Low-pH-induced conformational changes in the spike protein promote porcine deltacoronavirus infection by enhancing viral binding efficiency to the porcine aminopeptidase receptor

Abstract

ABSTRACT Porcine deltacoronavirus (PDCoV) is a critical enteric pathogen causing severe diarrhea in neonatal piglets, with fecal-oral transmission as its primary route. However, the molecular mechanism underlying PDCoV adaptation to the acidic gastric environment of hosts remains elusive. Here, we demonstrate that low-pH pretreatment (pH 4.0), mimicking the gastric niche of suckling piglets, significantly enhances PDCoV infectivity in porcine intestinal epithelial IPEC-J2 cells, as shown by elevated viral attachment, replication, and progeny titers. Accordingly, neutralization of gastric acid in piglets effectively attenuates PDCoV infection and pathogenicity in vivo. Mechanistically, acidic pH induces functionally important conformational rearrangements of the PDCoV spike (S) protein, which markedly increases its binding affinity for the cellular receptor porcine aminopeptidase N (pAPN), with the dissociation constant (KD) decreasing from 20.20 μM at pH 7.2 to 1.69 μM at pH 4.0. We solved the high-resolution cryo-electron microscopy (cryo-EM) structure of the PDCoV S protein under acidic conditions, representing the first deltacoronavirus S structure determined at low pH. Structural comparison with the neutral-pH closed conformation revealed that low pH drives a 14° outward rotation of the S1 C-terminal receptor-binding domain, reducing inter-domain interactions within the trimeric head and generating an intermediate activated conformation between the fully closed and fully open states, while the S2 fusion core remains structurally conserved. This pH-dependent conformational priming is shared by alpha- and deltacoronaviruses with intra-subunit S1 domain packing, which is distinct from the endosomal pH regulation of membrane fusion in betacoronaviruses, and represents an evolutionary adaptation to fecal-oral transmission. Our findings challenge the canonical view of gastric acid solely as a host defense barrier, uncover a preactivation role of the host physiological environment in viral infection, and provide new insights into enteric coronavirus biology, as well as novel targets for prevention and control strategies. IMPORTANCE Porcine deltacoronavirus (PDCoV), an emerging porcine enteric coronavirus, poses a serious threat to the swine industry and exhibits potential for cross-species transmission. This study demonstrated that the low-pH environment of the porcine stomach induces specific conformational changes in the PDCoV S protein, thereby maximizing the exposure of its receptor-binding domain, enhancing its binding to the host receptor, and promoting infection of intestinal epithelial cells. Our findings revealed that gastric acid functions not only as a physiological barrier but also as a key "molecular switch" that initiates PDCoV infection and is essential in enabling PDCoV to exploit the host physiological environment and complete its life cycle. These results provide new insights into the precise regulatory mechanisms governing the entry of PDCoV and other porcine enteric viruses, and establish a novel mechanistic basis for vaccine design, drug delivery, and formulation development. Porcine deltacoronavirus (PDCoV), an emerging porcine enteric coronavirus, poses a serious threat to the swine industry and exhibits potential for cross-species transmission. This study demonstrated that the low-pH environment of the porcine stomach induces specific conformational changes in the PDCoV S protein, thereby maximizing the exposure of its receptor-binding domain, enhancing its binding to the host receptor, and promoting infection of intestinal epithelial cells. Our findings revealed that gastric acid functions not only as a physiological barrier but also as a key "molecular switch" that initiates PDCoV infection and is essential in enabling PDCoV to exploit the host physiological environment and complete its life cycle. These results provide new insights into the precise regulatory mechanisms governing the entry of PDCoV and other porcine enteric viruses, and establish a novel mechanistic basis for vaccine design, drug delivery, and formulation development.