Discovery of 5' NAD capped viral RNAs reveals evolutionary divergent 5' metabolite capping across hepaciviruses

Hepatitis C virus (HCV), an important pathogen and prototype hepacivirus, 5' caps its RNA with flavin adenine dinucleotide (FAD); no other 5' metabolite capped viral RNAs have been identified. Here, we establish NADzymeID, allowing specific identification of 5' nicotinamide adenine dinucleotide (NAD). Using NADzymeID, we find that Norway rat hepacivirus 1 (NrHV-1) and related hepaciviruses use 5' NAD capping, whereas the closest relatives of HCV use 5' FAD, thereby revealing evolutionary divergence of 5' capping. NrHV-1 replication is attenuated in absence of NAD, and NrHV-1 is cleared more rapidly in mice with reduced intrahepatic NAD levels. As observed for 5' FAD, 5' NAD protects viral RNA from RIG-I mediated innate immune sensing. Thus, despite evolutionary divergence, metabolite capping generally confers evasion of innate immune responses. These represent the first examples of 5' NAD capping for viruses, providing tools also to investigate the elusive function of cellular 5' metabolite caps.