Cryo-EM structures of CCHFV polymerase reveal a stepwise initiation stabilization pathway and a dual-site inhibition mechanism.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a high-priority pathogen with high case-fatality rates, yet approved therapeutics remain unavailable. The CCHFV L segment encodes a ∼450-kDa RNA-dependent RNA polymerase (L protein) orchestrating viral replication, but its structural mechanisms remain elusive. Here, we present high-resolution cryo-EM structures of the CCHFV L protein in apo, 5' vRNA-bound, 5'/3' promoter-bound, and inhibitor-bound states. The catalytic core exhibits the canonical architecture of the Bunyavirales order, featuring conserved motifs and coordinated promoter recognition via a 5' vRNA "hook" and a secondary 3' vRNA-binding site. Using suramin as a probe, we identified a dual-site mechanism of polymerase inhibition. Suramin competitively occludes the 5' vRNA-binding pocket through electrostatic mimicry of the RNA backbone and concurrently traps a distal linker-fingers interface, restricting the conformational dynamics required for catalysis. Collectively, these findings provide structural insights into CCHFV polymerase regulation and inhibition.