Receptor affinity-selective differential dynamics of membrane fusion initiation govern deltacoronavirus cross-species transmission.

Porcine deltacoronavirus (PDCoV) exploits both human aminopeptidase N (hAPN) and porcine APN (pAPN) as receptors, with a higher affinity for hAPN than for pAPN through conserved interaction sites. However, despite this affinity, PDCoV is rarely pathogenic to humans, suggesting that the utilization dynamics of APN homologs by PDCoV are distinct, which is crucial in cross-species transmission but poorly understood. Here, we employed single-virus tracking to visualize and dissect the entry dynamics of PDCoV facilitated by APN. It was discovered that APN homologs bind PDCoV simultaneously, yet the times required for the initiation of membrane fusion and internalization differ significantly. Although high-affinity hAPN, rather than low-affinity pAPN, accompanies PDCoV during internalization, low-affinity pAPN initiates PDCoV internalization approximately 20 s faster than high-affinity hAPN, with caveolae-mediated endocytosis being more predominant and productive. Depending on the cell species, low-affinity pAPN induced a 5% to 25% greater proportion and a 0.6 to 4.3 min faster cell surface fusion, contributing to efficient infection. In contrast, high-affinity hAPN resulted in a 5% to 25% greater proportion and a 5 to 15 min faster endosomal fusion, potentially promoting immune evasion. We further demonstrated that the binding affinities between the PDCoV receptor-binding domain (RBD) and APN homologs are key determinants of the differential kinetics, driving flexible transitions between the two fusion pathways. This receptor affinity-selective PDCoV entry kinetics evolves an optimal balance of immune evasion and rapid infection, underscoring the potential for PDCoV interspecies transmission and the need for its vigilant surveillance.