RNA synthesis and substrate analog inhibition in the CCHFV polymerase

The about 4000-residue L proteins from the Nairoviridae are the largest known viral polymerases, lacking global structural information and promising nucleotide analog (NA) inhibitors. Here we report structures of full-length Nairoviridae Crimean-Congo hemorrhagic 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 RdRP active site and creating interaction networks critical for virus replication as suggested by CCHFV minigenome assay data. NAs with ribose-2′-modifications identical to the hepatitis C drug sofosbuvir are found to specifically and efficiently inhibit CCHFV RdRP through immediate chain termination mechanism. Using sofosbuvir-hepatitis C virus RdRP system as the reference, the potency of these NAs is further demonstrated in competition assays in the presence of corresponding NTPs.