RNASEK interacting with PEDV structural proteins facilitates virus entry via clathrin-mediated endocytosis

Porcine epidemic diarrhea virus (PEDV), as a type of Alphacoronavirus causing acute diarrhea and high death rate among sucking piglets, poses great financial damage to the swine industry. Nevertheless, the molecular mechanism whereby PEDV enters host cells is unclear, limiting the development of PED vaccines and anti-PEDV agents. The present study found that the host protein ribonuclease kappa (RNASEK) was regulated by USF2, a transcription factor, and facilitated the PEDV replication. RNASEK was identified as a novel binding partner of PEDV, which interacted with a spike (S), envelope (E), and membrane (M) proteins on PEDV virion surfaces to increase the uptake not for attachment of PEDV virions. PEDV enters cells through the endocytosis pathways. RNASEK knockdown or RNASEK knockout assay revealed that through clathrin-mediated endocytosis (CME), RNASEK promoted the internalization of PEDV virions. Clathrin and the adaptor protein EPS15 only interacted with PEDV E protein, demonstrating that the RNASEK could target more virions through interaction with PEDV S, E, and M proteins to clathrin and EPS15 proteins rather than merely interacting with PEDV E protein to mediate the PEDV entry through CME. Moreover, our findings suggest that RNASEK, a newly identified host-entry factor, facilitates PEDV internalization by increasing the interaction of PEDV virions and EPS15-clathrin complex and may also provide a potential target for anti-PEDV therapies.IMPORTANCEPEDV is the causative pathogen of porcine diarrhea, which is a highly infectious acute intestinal condition, that poses significant economic damage to the swine industry. However, the existing PED vaccines fail to provide adequate protection for piglets against PEDV infection. Although PEDV replication in cells has been widely described, the mechanisms beneath PEDV entry of the host cells are incompletely understood. In this study, we showed that RNASEK, regulated by the transcription factor USF2, is a new host factor increasing PEDV infection in LLC-PK1 cells. RNASEK can bind to multiple structural proteins of PEDV (S, E, and M proteins), therefore increasing the interaction between PEDV virions, clathrin, and EPS15 to promote PEDV virion entry. Apart from unraveling the entry mechanisms of PEDV, our findings also contributed to facilitating the development of anti-PEDV agents and PED vaccines.