Revisiting the roles of trypsin in the productive infection of porcine deltacoronavirus in porcine-derived cells

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus with the potential for interspecies transmission. Trypsin has been shown to play a positive role in the isolation and multiplication of PDCoV in vitro, however, the functions of trypsin during PDCoV replication cycle remain controversial. In this study, we revisited the roles of trypsin for PDCoV infection by utilizing two kinds of PDCoV, PDCoV^T+ and PDCoV^T−, which were prepared in the presence or absence of trypsin, respectively. We found that PDCoV^T+ was able to continuously proliferate in the medium containing trypsin, achieving a higher titer as the infection progress in LLC-PK1 and other tested porcine-derived cells. However, its replication was only transiently improved at 12 hours post-infection, and lower viral titers were observed under trypsin-free culture conditions. Furthermore, the trypsin-mediated enhancement of viral replication could be inhibited by trypsin inhibitor SBTI, suggesting that the second-round viral reproduction of PDCoV^T+ might be impeded without trypsin. We further investigated the replication dynamics of PDCoV^T− in LLC-PK1 cells in the presence or absence of trypsin. The results indicated that PDCoV^T− generated lower viral titers under trypsin-free culture conditions, while the addition of trypsin reverted the infectivity of PDCoV^T−. Additionally, we demonstrated that trypsin cleaved the PDCoV spike protein, activating viral attachment and internalization. Moreover, trypsin promoted viral replication and release, accelerating PDCoV maturation and facilitating second-round infection. Taken together, this study systematically revaluated and emphasized an essential role of trypsin in PDCoV infection, providing mechanistic insights into the productive infection of PDCoV in porcine-derived cells.