We recently discovered that hepatitis C virus (HCV) is capped with flavin adenine dinucleotide (FAD) on the 5’ end of its RNA and showed that the RNA-dependent RNA polymerase NS5B is responsible for its incorporation as a non-canonical initiating nucleotide (NCIN). Thus, to elucidate the structural basis of FAD-initiated HCV replication and to shed light on the poorly understood de novo initiation step, we used X-ray crystallography to determine structures of NS5B in complex with RNA and FAD.

Obtaining structures of NS5B with RNA bound is challenging and was previously only possible using a mutated genotype 2a NS5B (strain JFH1) containing five substitutions of which S15G/C223H/V321I are near the active site and therefore could influence substrate recognition. Here, we successfully obtained well-diffracting crystals capable of binding RNA and FAD by using only two protein surface substitutions, thus enabling structural studies in a near-wild-type context. To further enhance physiological relevance, we used the authentic NTP substrate in the active site rather than the commonly used NDP model substrate.

We successfully obtained de novo initiation structures of NS5B-RNA-FAD at high resolutions ranging from 1.7 to 2.2 Å. We found that FAD binding is stabilized by key interactions with Y448 and G449 in NS5B’s beta-loop, as well as stacking and base pairing with the RNA. The structures reveal that the authentic NTP substrate promotes catalytically competent conformations of FAD and the incoming nucleotide. Additionally, we observed structural consequences of S15G/C223H/V321I, including altered active site electrostatics and changes in NS5B–RNA interactions.

Overall, our structures provide new insights into HCV replication, elucidate the role of non-canonical initiating nucleotides in viral RNA synthesis and establish a platform for studying de novo initiation, including nucleotide analog recognition in a physiologically relevant NS5B context.