An Da, Li Yan, Pei Yi, Ma Yong-Hao, Yao Fei, Feng Yongzhi, Shan Yanke, Liu Fei
Joint International Research Laboratory of Animal Health and Food Safety of Ministry of Education, Single Molecule Biochemistry & Biomedicine Laboratory (Sinmolab), Nanjing Agricultural University, Nanjing, Jiangsu, China.
Sanya Institute of Nanjing Agricultural University, Sanya, Hainan, China.
PLoS Pathog. 2025 Jun 30;21(6):e1013317. doi: 10.1371/journal.ppat.1013317. eCollection 2025 Jun.
Aminopeptidase N (APN) plays multiple roles in various physiological processes, with its function as a viral receptor in several coronaviruses being one of the most prominent. However, the role of porcine APN (pAPN) in porcine epidemic diarrhea virus (PEDV) has remained controversial. Single-virus tracking enables a more comprehensive dynamic dissection of pAPN utilization during virus entry. In this study, a comparative analysis of pAPN usage by PEDV, transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) provides more precise and quantitative insights into pAPN's specific role in PEDV entry. Here, we used molecular docking and surface plasmon resonance (SPR) to demonstrate that pAPN binds to PEDV, with lower affinity than to TGEV. However, pAPN facilitates PEDV replication through internalization only in susceptible cells, not in non-susceptible cells. Using single-virus tracking, we observed that pAPN triggers PEDV internalization via clathrin- and caveolae-mediated endocytosis, resembling a receptor-mediated process. pAPN participates in 35% of PEDV internalization events, but mediates 80% of TGEV internalization, with pAPN-mediated PEDV internalization occurring approximately 60 s slower than TGEV. The dynamic differences in the internalization of PEDV and TGEV mediated by pAPN primarily arise during the binding stage prior to the initiation of accelerated directional movement, whereas their durations of movement are comparable. Additionally, we found that the internalization dynamics of porcine deltacoronavirus (PDCoV), which also uses pAPN as a receptor, are similar to those of TGEV. These findings resolve the controversy surrounding pAPN's role in PEDV entry, and highlight the dynamic differences in PEDV, TGEV, PDCoV, and SADS-CoV internalization via pAPN at single-virus level, providing a novel theoretical basis for the potential receptor evaluation from kinetic perspective, which could significantly contribute to the development of strategies against future PEDV outbreaks.
氨肽酶N（APN）在多种生理过程中发挥多种作用，其中它作为几种冠状病毒的病毒受体的功能最为突出。然而，猪氨肽酶N（pAPN）在猪流行性腹泻病毒（PEDV）中的作用一直存在争议。单病毒追踪能够更全面地动态剖析病毒进入过程中pAPN的利用情况。在本研究中，对PEDV、传染性胃肠炎病毒（TGEV）和猪急性腹泻综合征冠状病毒（SADS-CoV）对pAPN的使用情况进行比较分析，能更精确和定量地洞察pAPN在PEDV进入过程中的具体作用。在此，我们使用分子对接和表面等离子体共振（SPR）证明pAPN与PEDV结合，但其亲和力低于与TGEV的结合。然而，pAPN仅在易感细胞中通过内化促进PEDV复制，在非易感细胞中则不然。通过单病毒追踪，我们观察到pAPN通过网格蛋白和小窝介导的内吞作用触发PEDV内化，类似于受体介导的过程。pAPN参与了35%的PEDV内化事件，但介导了80%的TGEV内化，pAPN介导的PEDV内化比TGEV慢约60秒。pAPN介导的PEDV和TGEV内化的动态差异主要出现在加速定向运动开始之前的结合阶段，而它们的运动持续时间相当。此外，我们发现同样以pAPN为受体的猪德尔塔冠状病毒（PDCoV）的内化动态与TGEV相似。这些发现解决了围绕pAPN在PEDV进入中作用的争议，并突出了PEDV、TGEV、PDCoV和SADS-CoV在单病毒水平上通过pAPN内化的动态差异，为从动力学角度评估潜在受体提供了新的理论基础，这可能对未来PEDV疫情防控策略的制定有重大贡献。
