RNA synthesis and substrate analogue inhibition in the CCHFV polymerase

The about 4,000-residue L proteins from the Nairoviridae are the largest known viral polymerases, lacking global structural information and promising nucleotide analogue inhibitors. Here we report structures of full-length Nairoviridae Crimean-Congo haemorrhagic fever virus (CCHFV) L, including a 3.0 Å resolution polymerase elongation complex that elucidates mechanisms of both early and late elongation stages. Large additions and insertions are found in all three major functional regions that contain the endonuclease RNA-dependent RNA polymerase (RdRP) and cap-binding domain of CCHFV L, extending RNA-binding paths on both sides of the RdRP active site and creating interaction networks critical for viral replication, as suggested by CCHFV minigenome assay data. Nucleotide analogues with ribose-2' modifications identical to the hepatitis C drug sofosbuvir are found to specifically and efficiently inhibit CCHFV RdRP through an immediate chain termination mechanism. Using a sofosbuvir-hepatitis C virus RdRP system as the reference, the potency of these nucleotide analogues is further demonstrated in competition assays in the presence of corresponding nucleoside triphosphates.