Porcine deltacoronavirus accessory protein NS7a possesses the functional characteristics of a viroporin

Viroporins are virus-encoded proteins that mediate ion channel (IC) activity, playing critical roles in virus entry, replication, pathogenesis, and immune evasion. Previous studies have shown that some coronavirus accessory proteins have viroporin-like activity. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that encodes three accessory proteins, NS6, NS7, and NS7a. However, whether any of the PDCoV accessory proteins possess viroporin-like activity, and if so which, remains unknown. In this study, we analyzed the biochemical properties of the three PDCoV-encoded accessory proteins and found that NS7a could enhance the membrane permeability of both mammalian cells and Escherichia coli cells. Indirect immunofluorescence assay and co-immunoprecipitation assay results further indicated that NS7a is an integral membrane protein and can form homo-oligomers. A bioinformation analysis revealed that a putative viroporin domain (VPD) is located within amino acids 69-88 (aa69-88) of NS7a. Experiments with truncated mutants and alanine scanning mutagenesis additionally demonstrated that the amino acid residues ⁶⁹FLR⁷¹ of NS7a are essential for its viroporin-like activity. Together, our findings are the first to demonstrate that PDCoV NS7a possesses viroporin-like activity and identify its key amino acid residues associated with viroporin-like activity.