The aim of this project is to structurally and functionally characterise the influenza virus RNA polymerase complex that transcribes and replicates the influenza virus RNA genome. Structural and functional data about the RNA polymerase and its interaction with cellular factors will greatly expand our limited knowledge of the mechanisms of influenza virus genome transcription and replication.
The influenza virus genome consists of eight single stranded negative-sense RNAs that form viral ribonucleoprotein (vRNP) complexes with the viral RNA polymerase and oligomeric nucleoprotein (NP). vRNPs display a double-helical arrangement resembling a large loop twisted into a helical filament. The RNA polymerase, a 250-kDa heterotrimeric complex, containing the polymerase basic 1 (PB1), polymerase basic 2 (PB2), and polymerase acidic (PA) subunits, binds at one end of the filament interacting with both the 5ʹ and the 3ʹ ends of the vRNA. The viral polymerase is responsible for both transcribing and replicating the viral RNA genome within the RNP in the nucleus of the infected cell. Transcription and replication require different initiation and termination strategies but the control mechanisms involved and the contribution of cellular factors remain poorly characterised at the molecular level.
Recently, we developed methods to express and purify the heterotrimeric influenza virus RNA polymerase using a baculovirus technology and determined the structure of the influenza C virus polymerase using x-ray crystallography (see Figure). This revealed the polymerase in a novel, transcriptionally inactive conformation. However, it is currently unknown how the polymerase is activated, in order to initiate primer-dependent transcription or primer-independent replication. We aim to determine the structure of the transcribing and replicating polymerase, using a combination of x-ray crystallography and cryo-EM, in conformations representing the initiating, elongating and terminating polymerases. We also aim to determine the structure of the RNA polymerase bound to cellular factors, i.e. the C-terminal domain (CTD) of RNA polymerase II (hypothesised to promote viral transcription) and importin 5 (involved in the nuclear import of the PB1-PA polymerase dimer).