Cathepsin L and transmembrane serine protease 11E mediate trypsin-independent entry of porcine deltacoronavirus into Huh7 cells.

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.