HCV NS3/4A protease relocalizes CCTα to viral replication sites, enhancing phosphatidylcholine synthesis and viral replication.

Positive-sense single-stranded RNA [(+)RNA] viruses constitute more than one-third of all virus genera, including numerous pathogens of clinical significance. All (+)RNA viruses reorganize cellular membranes from organelles to establish replication compartments (RCs). These RCs are thought to form a platform for membrane-associated replicases, in addition to protecting the viral RNAs from cytosolic innate immune signaling and RNA-degradation machinery. Previous work demonstrated that three families of (+)RNA viruses, namely , , and , commonly induce the accumulation of phosphatidylcholine (PC) at their RCs. This phenomenon suggests a potential avenue for a broad-spectrum antiviral strategy targeting PC metabolism. Our study elucidates three key observations: i) hepatitis C virus (HCV) infection prompts the relocalization of CCTα, the rate-limiting enzyme in PC synthesis, to the RCs; ii) the enhancement of PC synthesis is contingent upon the protease activity of the NS3/4A protein; and iii) utilizing click chemistry, we demonstrate that HCV infection stimulates de novo PC synthesis at the viral replication site through the Kennedy pathway. These findings provide significant insights into the manipulation of lipid metabolism by HCV during RC formation, a mechanism likely conserved across various (+)RNA virus families.