N-linked glycoproteins and host proteases are involved in swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is highly pathogenic to piglets and poses a major threat to the swine industry. SADS-CoV has a wide cell tropism and pathogenic potential in younger animals. Therefore, understanding how SADS-CoV enters cells is essential for curbing its re-emergence and spread. Here, we report that tunicamycin, an N-linked glycoprotein inhibitor, inhibited the attachment of SADS-CoV to host cells, suggesting that the SADS-CoV receptor may be an N-linked glycoprotein but not Neu5Gc or Neu5Ac. Moreover, we found that exogenous trypsin, endogenous serine protease, cathepsin B, cathepsin L, and lysosomal acidification triggered SADS-CoV entry into cells. These findings improve our understanding of the molecular mechanisms underlying SADS-CoV entry and provide insights into the development of potential antiviral targets against SADS-CoV.IMPORTANCEGaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.