Uridine cytidine kinase 1 promotes phosphatidylcholine biosynthesis, facilitating SARS-CoV-2 infection.

RNA viruses require diverse metabolic intermediates for replication, including nucleotides, which are synthesized through two pathways: de novo biosynthesis and salvage. De novo nucleotide biosynthesis is required for viral replication, but the role of nucleotide salvage is less clear. Genetic screening of purine and pyrimidine salvage pathways revealed a requirement for pyrimidine salvage in SARS-CoV-2 viral replication. Although both UCK1 and UCK2 catalyze the rate-limiting monophosphorylation of pyrimidines for pyrimidine salvage, we show that UCK2 is the major enzyme for cytoplasmic salvage of extracellular pyrimidines, functioning redundantly with de novo biosynthesis for RNA replication, and is thus dispensable for SARS-CoV-2 infection under conditions where de novo biosynthesis is active. In contrast, we find that UCK1 supports SARS-CoV-2 replication by specifically promoting nuclear cytidine triphosphate (CTP) synthesis for phosphatidylcholine (PC) biosynthesis, regulating peroxisomal lipid metabolism, and facilitating organelle interactions with double-membrane vesicles (DMVs), lipid structures required for SARS-CoV-2 RNA replication.