De novo initiation of RNA synthesis by a recombinant flaviridae RNA-dependent RNA polymerase

Recombinant RNA-dependent RNA polymerases have been reported to synthesize RNAs by extending from the 3' hydroxyl of a template or an oligonucleotide primer. De novo initiation has not been reported. Establishment of such an assay would facilitate the analysis of the initiation requirements and allow the testing of antiviral compounds specifically targeting initiation. Using chemically synthesized RNAs and DNAs, we demonstrate that the recombinant RNA-dependent RNA polymerase (NS5B) of bovine viral diarrhea virus initiates de novo RNA synthesis. Nucleotides required for efficient initiation of RNA synthesis and for stable interaction with NS5B were identified. (C) 1999 Academic Press.