biostudies-literatureSherwood AVEuropean Research CouncilNovo Nordisk Fonden811-818https://www.ebi.ac.uk/biostudies/studies/S-EPMC7616780biostudies-literatureUnknown619(7971)RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus^1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer³. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life^4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.NatureHepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.PMC7616780NNF18OC0055462NNF18OC0054518802899NNF19OC0058443NNF18OC0052354NNF22SA0078213Rivera-Rangel LRLangvad NWAnker KMJakljevic EFossat NAl-Chaer AScheel TKHPham LVFernandez-Antunez CKusnierczyk ASolund CPodolska-Charlery ANielsen LBottaro SSherwood AVRyberg LAGottwein JMLarsen HSOffersgaard AVinther JRamirez SVagbo CBHolmbeck KGrothen JERCosta RWeis NBukh JIndrisiunaite GfalseHepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus^1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer³. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life^4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.2023-01-01T00:00:00Z2023 Jul2026-06-02T11:37:18.153Z2025-04-21T21:50:01.124ZS-EPMC76167803740781710.1038/s41586-023-06301-3