Transcription, replication, trafficking and assembly of the influenza virus RNA genome

Influenza viruses are important human and animal pathogens that have the potential to cause severe respiratory disease and death in humans. Influenza A and B viruses are responsible for seasonal epidemics and influenza A viruses also have the potential to cause pandemics. Influenza A viruses infect a wide range of animal species, with wild waterfowl being considered their natural reservoir. All four influenza A virus pandemics of recent human history occurred when either an avian influenza virus transmitted directly into the human population, giving rise to the 1918 pandemic, or avian or swine influenza viruses combined with human seasonal influenza virus, leading to the 1957, 1968 and 2009 pandemics. In the case of a new emerging influenza pandemic virus, no vaccines would be available initially and antivirals would be crucial to tackle the first wave of the virus; however, currently available influenza antivirals are limited.

The focus of our research programme is the RNA polymerase of influenza viruses, a large multifunctional enzyme complex. The function of the influenza virus RNA polymerase is to make copies of the genetic information of the virus, stored in segments of RNA. After an influenza virus infects a cell, the RNA polymerase first makes copies of the genetic material, which the cell is then 'forced' to read to make viral proteins, building blocks of new virus particles. The RNA polymerase also makes copies of the genetic material to be packed into new viruses and recent research suggests that it even plays a role in transporting the new genetic material to the sites in the cells where new virus particles are assembled. The RNA polymerase of influenza viruses is considered a prime viral target for the development of novel antiviral drugs; without it the virus is unable to make new copies of itself and spread to infect further hosts. Indeed, several polymerase inhibitors have been developed recently which are licensed for use in emergency in a limited number of countries. However, development of resistance against these antivirals has already been documented.

Our research programme aims to understand exactly how the influenza virus RNA polymerase reprogrammes the cell to make viral proteins and produce new copies of the genetic material to be packed into new virus particles. To perform these functions the virus hijacks host proteins that act together with the viral RNA polymerase. We aim to understand how the viral RNA polymerase interacts with these host proteins, how these host proteins assist the RNA polymerase in promoting the copying of the viral genetic material, and where these interactions occur in the host cell. We also aim to elucidate the role of the viral RNA polymerase in transporting the copied genetic material to the sites of virus assembly inside the host cell. Addressing these objectives will result in new knowledge about the fundamental biology of influenza virus infections. As such, it will also help with the assessment of the pandemic potential of emerging influenza virus strains and therefore has potential to inform public health policies. Importantly, results from this programme will underpin further work into targeting the viral RNA polymerase for antiviral development and could contribute to the development of improved or entirely novel influenza vaccines. To translate findings from our current and proposed research into antiviral therapies, we already have established links with industrial partners. Furthermore, we actively seek new opportunities with industrial collaborators for translating the knowledge generated.

Our research programme focuses on the RNA-dependent RNA polymerase of influenza viruses. It aims to uncover the molecular mechanisms of viral transcription by the influenza virus polymerase in the context of interactions with the host RNA polymerase II transcriptional machinery. It also aims to provide a mechanistic understanding of influenza virus genome replication, co-replicative assembly of nascent viral RNA into a ribonucleoprotein complex and the involvement of the ANP32 family of host proteins. We will specifically address the functional significance of avian to mammalian adaptive mutations in the influenza A virus polymerase in the context of dimers of polymerase heterotrimers and identify and characterise sites of transcription and replication in the nucleus. Furthermore, our programme aims to uncover the mechanisms governing the nuclear export, cytoplasmic transport and assembly of the multi-segmented influenza virus RNA genome, processes in which the viral RNA polymerase plays a central role.

To meet these objectives we will use a cross-disciplinary approach, combining biochemical, biophysical and virological methods, including reverse genetics for generating influenza virus mutants, proximity labelling based on TurboID and APEX and mass spectrometry, cryo-electron microscopy, x-ray crystallography, small angle light scattering (SAXS), super-resolution microscopy, next generation sequencing, CRISPR/Ca9 genome editing and bioinformatics. Although this is fundamental research primarily aimed at improving our understanding of the biology of influenza virus infections, we will fully exploit its potential for developing novel antivirals, improved vaccines and informing public health policies. To this end, we will continue our already established collaborations with industrial partners and seek further collaborations; any new discoveries will be fully exploited through University of Oxford technology transfer routes.