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).
Molecular and cell biology, virology, structural biology, x-ray crystallography, cryo-electron microscopy, small angle X-ray scattering, biophysical characterisation
Project reference number: 746
|Professor Jonathan M Grimes||Structural Biology||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRfirstname.lastname@example.org|
|Professor Ervin Fodor||Dunn School of Pathology||University of Oxford||GBRemail@example.com|
Negative-strand RNA viruses represent a significant class of important pathogens that cause substantial morbidity and mortality in human and animal hosts worldwide. A defining feature of these viruses is that their single-stranded RNA genomes are of opposite polarity to messenger RNA and are replicated through a positive-sense intermediate. The replicative intermediate is thought to exist as a complementary ribonucleoprotein (cRNP) complex. However, isolation of such complexes from infected cells has never been accomplished. Here we report the development of an RNA-based affinity-purification strategy for the isolation of cRNPs of influenza A virus from infected cells. This technological advance enabled the structural and functional characterization of this elusive but essential component of the viral RNA replication machine. The cRNP exhibits a filamentous double-helical organization with defined termini, containing the viral RNA-dependent RNA polymerase (RdRp) at one end and a loop structure at the other end. In vitro characterization of cRNP activity yielded mechanistic insights into the workings of this RNA synthesis machine. In particular, we found that cRNPs show activity in vitro only in the presence of added RdRp. Intriguingly, a replication-inactive RdRp mutant was also able to activate cRNP-templated viral RNA synthesis. We propose a model of influenza virus genome replication that relies on the trans-activation of the cRNP-associated RdRp. The described purification strategy should be applicable to other negative-strand RNA viruses and will promote studies into their replication mechanisms. Hide abstract