Proteases facilitate the endosomal escape of porcine epidemic diarrhea virus during entry into host cells

•The endosomal escapes of PEDV during the entry occur in 4 h after virus inoculation in the presence of exogenous proteases in the medium.•Without proteases in the medium, PEDV remained in the endosomes and degraded.•Inhibitors of endosome maturation or cathepsin B/L blocked PEDV entry by retaining viruses in the endosomes. Exogenous and endogenous proteases play important roles in porcine epidemic diarrhea virus (PEDV) entry and replication. The roles of proteases in the viral endosomal escape and replication using trypsin (KD) or elastase (AA)-adapted US PEDV strains were studied. While PEDV KD and AA require different exogenous protease for efficient replication in cells, PEDV KD was more dependent on the protease than PEDV AA. There was no marked difference in viral trafficking between them during the entry events. Both PEDV were observed in the endosomes with or without protease at 1 h after virus inoculation. With protease, viral signals in the endosomes disappeared after 4 h, and newly synthesized viral proteins were detected in the ER after 6 h. However, without protease, viruses remained in the endosomes up to 24 h, which correlated with limited virus replication. Inhibitors of cathepsins, endogenous proteases, significantly reduced the replication of both PEDV by interfering with the viral endosomal escape.