Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter

The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction.