Xiao Wenwen, Xiong Yuanxiang, Wang Yuchen, Li Ting, Chen Chaoqun, Shi Yuting, Su Guanning, Zhou Yanrong, Xiao Shaobo, Fang Liurong
National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.
J Virol. 2025 Aug 11:e0105525. doi: 10.1128/jvi.01055-25.
Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus with zoonotic potential. Previous studies showed that PDCoV productive infection is dependent on exogenous trypsin in porcine-derived cells. In this study, we found that trypsin appears to be dispensable for PDCoV infection in human-derived cell lines. Using Huh7 cells as a model, we investigated the potential mechanisms underlying this phenomenon. Our results demonstrated that exogenous trypsin has no significant impact on PDCoV proliferation in human-derived cells. Furthermore, the culture supernatants collected from Huh7 cells can promote PDCoV infection in porcine-derived cells under trypsin-free conditions. By utilizing protease inhibitors, we identified that PDCoV enters Huh7 cells via endosomal and plasma membrane fusion pathways. Further investigations revealed that cathepsin L (CTSL) and transmembrane serine protease 11E (TMPRSS11E) mediate these pathways, respectively. Mechanistically, CTSL and TMPRSS11E cleave the PDCoV spike (S) protein, activating membrane fusion and promoting viral entry into Huh7 cells. Additionally, our results indicated that the highly expressed furin in Huh7 cells also plays a critical role in the late stages of PDCoV replication cycle, facilitating virion maturation and release. Taken together, these findings elucidate the detailed mechanisms of PDCoV infection in human-derived cells, highlighting the critical roles of CTSL and TMPRSS11E in trypsin-independent entry.
PDCoV can be isolated from human plasma samples and infect various human-derived cells, raising significant concerns regarding its potential for cross-species transmission. Coronavirus invasion involves receptor binding and spike (S) protein cleavage by proteases. While human aminopeptidase N (APN) has been confirmed as a receptor that mediates PDCoV infection, the specific proteases involved in infections of human-derived cells remain incompletely understood. Here, we investigated the mechanisms by which PDCoV enters human-derived cells and demonstrated that PDCoV infection in these cells is independent of exogenous trypsin. Furthermore, we identified two critical proteases, CTSL and TMPRSS11E, which facilitate PDCoV entry into Huh7 cells via endosomal and plasma membrane fusion pathways, respectively. Additionally, we discovered that furin promotes the maturation and release of virions. This study reveals the infection mechanisms of PDCoV in human-derived cells, highlighting the roles of CTSL, TMPRSS11E, and furin in viral entry and release.
猪德尔塔冠状病毒（PDCoV）是一种具有人畜共患病潜力的新兴肠道冠状病毒。先前的研究表明，PDCoV的有效感染依赖于猪源细胞中的外源性胰蛋白酶。在本研究中，我们发现胰蛋白酶对于人源细胞系中的PDCoV感染似乎是可有可无的。以Huh7细胞为模型，我们研究了这一现象背后的潜在机制。我们的结果表明，外源性胰蛋白酶对人源细胞中PDCoV的增殖没有显著影响。此外，从Huh7细胞收集的培养上清液在无胰蛋白酶条件下可促进猪源细胞中的PDCoV感染。通过使用蛋白酶抑制剂，我们确定PDCoV通过内体和质膜融合途径进入Huh7细胞。进一步的研究表明，组织蛋白酶L（CTSL）和跨膜丝氨酸蛋白酶11E（TMPRSS11E）分别介导这些途径。从机制上讲，CTSL和TMPRSS11E切割PDCoV刺突（S）蛋白，激活膜融合并促进病毒进入Huh7细胞。此外，我们的结果表明，Huh7细胞中高表达的弗林蛋白酶在PDCoV复制周期的后期也起着关键作用，促进病毒粒子的成熟和释放。综上所述，这些发现阐明了PDCoV在人源细胞中的详细感染机制，突出了CTSL和TMPRSS11E在不依赖胰蛋白酶的进入过程中的关键作用。
PDCoV可从人血浆样本中分离出来并感染各种人源细胞，这引发了人们对其跨物种传播潜力的重大担忧。冠状病毒的入侵涉及受体结合和蛋白酶对刺突（S）蛋白的切割。虽然人氨肽酶N（APN）已被确认为介导PDCoV感染的受体，但参与人源细胞感染的具体蛋白酶仍不完全清楚。在这里，我们研究了PDCoV进入人源细胞的机制，并证明PDCoV在这些细胞中的感染不依赖于外源性胰蛋白酶。此外，我们确定了两种关键蛋白酶，CTSL和TMPRSS11E，它们分别通过内体和质膜融合途径促进PDCoV进入Huh7细胞。此外，我们发现弗林蛋白酶促进病毒粒子的成熟和释放。这项研究揭示了PDCoV在人源细胞中的感染机制，突出了CTSL、TMPRSS11E和弗林蛋白酶在病毒进入和释放中的作用。
