Membrane protein CRISPR screen identifies RPSA as an essential host factor for porcine epidemic diarrhea virus replication

ABSTRACT Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. The host factors required for PEDV replication, including receptors, remain poorly understood. Here, we developed a porcine membrane-protein-scale CRISPR/Cas9 knockout (PigMpCKO) library and performed two rounds of PEDV infection. Ribosomal protein SA (RPSA), the known receptor of dengue virus, was found to be a potent host factor. Moreover, our research revealed that RPSA is involved in the replication stage of PEDV and not in the entry stage. Inhibitor and activator experiments demonstrated that knockout (KO) of RPSA downregulates the ERK1/2 signaling pathway to impair PEDV infection. Additionally, RNA sequencing data indicated that cellular lipid biosynthesis and lipid transport processes were significantly inhibited in the absence of RPSA during PEDV infection. Mechanistic studies revealed that the reduction in total cholesterol and triglyceride levels, resulting from RPSA KO, was partially mediated by the ERK1/2 pathway, leading to impaired lipid accumulation during PEDV replication. Interestingly, RPSA KO also significantly downregulated the expression of aminopeptidase N (APN) and inhibited infection by transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), both of which belong to the swine enteric coronavirus group. In summary, our results establish RPSA as a novel host factor that is critical for coronavirus replication. This provides new insights into the mechanisms of virus-host interactions and paves the way for the development of broad-spectrum antiviral therapies. IMPORTANCE Swine enteric coronaviruses (SeCoVs) cause severe economic losses to the global swine industry and pose a potential threat to public health. Identification of receptors required for PEDV infection could develop novel targets for drug therapy and disease-resistant breeding. We conducted a CRISPR/Cas9 screen targeting membrane proteins in porcine kidney cells infected with PEDV to identify possible receptors and discovered numerous novel candidate host factors. Considering RPSA’s known role as a receptor for multiple viruses, we focused on investigating its potential in coronavirus infection. Our results revealed that RPSA does not contribute to the entry stage but to the replication stage of coronavirus infection. We first reported the role that RPSA plays in the regulation of APN expression and lipid metabolism. RPSA is essential for PEDV and other SeCoVs replication, providing a novel insight into the search for the receptor of PEDV and identifying potential therapeutic targets for coronaviruses.