An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.

Abstract

Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the 'promoter'). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.

• Authors: Stefan Reich, Delphine Guilligay, Stephen Cusack
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 45 Issue 6 Pg. 3353-3368 (04 07 2017) ISSN: 1362-4962 [Electronic] England
• PMID: 28126917 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Chemical References

• RNA, Viral
• Viral Proteins
• DNA-Directed RNA Polymerases

Topics

• Base Pairing
• DNA-Directed RNA Polymerases (antagonists & inhibitors, chemistry, metabolism)
• Fluorescence Polarization
• Influenza B virus (enzymology, genetics, physiology)
• Promoter Regions, Genetic
• RNA, Viral (biosynthesis, chemistry)
• Transcription, Genetic
• Viral Proteins (chemistry, metabolism)
• Virus Replication