Hepatitis C virus RNA is 5' flavin adenine dinucleotide (FAD) capped

RNA viruses have developed elaborate strategies for 5' capping and protection of their genomes. However, so far no 5' RNA cap has been identified for Hepatitis C virus (HCV), which cause chronic infection, liver cirrhosis and cancer in humans4. Here, we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as noncanonical initiating nucleotide by the viral RNA-dependent RNA polymerase resulting in a 5' FAD cap on the HCV RNA. FAD capping is completely conserved for the HCV replication intermediate negative RNA strand and partially for the positive RNA strand. The prototype strain J6/JFH1 RNA is FAD capped when isolated from the liver and serum of a human liver chimeric mouse model and in vitro the FAD capping frequency is ~75 %, which is the highest metabolite RNA capping frequency observed. Furthermore, we show that 5' FAD capping protects RNA from cell-intrinsic innate immune recognition but has limited effect on HCV RNA stability. These results establish capping with cellular metabolites, such as FAD, as a novel viral RNA capping and immune evasion strategy, which potentially could be used by many viruses for protection of their intermediate RNA strands and thereby affect viral treatment outcomes and persistency. Overall design: Total RNA was isolated from cells infected with different viruses. CapZyme-seq protocol was applied to total RNA or RNA treated with TEX enzyme. RNA was enzymatically treated with de-capping enzymes that specifically convert different 5' cap structures to 5' monophosphates. 5'-phosphate dependent adapter ligation was performed followed by reverse transcription. Libraries were generated using PCR with custom Illumina compatible primers. For experiments with HCV genotype 2a J6/JFH1-clone2, 5 biological replicates with 2 technical replicates per each biological replicate were analyzed for each enzymatic treatment condition (NUDX23, Rpp and no enzyme control). For the experiments with other HCV strains, 1 biological replicate was analyzed for each enzymatic condition. For the experiments with BVDV, 3 biological replicates were analyzed for each enzymatic conditions. For the experiments with CHKV, 2 biological replicates were analyzed for each enzymatic conditions. For the experiments with TBEV, 3 biological replicates were analyzed for each NUDX23, Rpp and no enzyme control enzymatic conditions and 3 biological replicates were analyzed for each NudC, Rpp and no enzyme control enzymatic conditions.