Non-segmented negative-strand RNA viruses (nsNSVs) including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses can cause respiratory infections, hemorrhagic fever and encephalitis in the humans and animals, and are considered as substantial health and economic burden worldwide1. Replication and transcription of the viral genome are executed by the large L polymerase that is a promising target for the development of antiviral drugs. Here, using EBOV L polymerase as a representative, we showed that de novo replication of L polymerase is controlled by the specific 3' leader sequence of EBOV genome in an enzymatic assay, and formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence. We then determined the high-resolution structures of EBOV L-VP35-RNA complex and found that the 3' leader RNA binds in the template entry channel with a distinctive stable bend conformation. Further mutagenesis work confirmed that the bend conformation of RNA is required for the de novo replication activity and revealed the key residues of L protein that stabilize the RNA conformation. These findings have provided a new mechanistic understanding of RNA synthesis for nsNSV polymerases, and revealed important targets for the development of antiviral drugs.
© 2023. The Author(s), under exclusive licence to Springer Nature Limited.