Structure-function relationships of the influenza virus RNA polymerase: influence on virulence, host restriction and innate immune responses

Seasonal influenza viruses cause 3 to 5 million cases of severe infections in humans each year, leading to between 250,000 to 500,000 deaths worldwide. Influenza A viruses can also cause pandemics, through the emergence of new viral strains from wild birds and pigs, with potentially catastrophic consequences for society. Currently the H5N1 and H7N9 'bird flu' subtypes are considered the biggest threats as these are prevalent in birds and can infect humans directly, leading to severe disease and death. The concern is that these viruses, or other 'bird flu' subtypes, through adaptation or by mixing with human or swine influenza viruses, could gain the ability to easily transmit between humans and lead to a new pandemic. Current antiviral approaches are limited to vaccines that require annual updating and are unlikely to be useful against an emerging novel pandemic virus, and a few antiviral drugs against which viral resistance can develop. The focus of our research programme is the RNA polymerase of influenza viruses, a key multi-subunit enzyme complex containing multiple functional domains, considered to be a prime viral target for the development of novel antiviral drugs.
The viral RNA polymerase is responsible for copying the genetic information of influenza virus that is stored in eight segments of RNA. It is directly responsible for transcribing the RNA segments into instructions to the host cell to produce viral proteins (messenger mRNA, mRNA) as well as replicating the RNA segments through complementary RNA intermediates. The viral proteins together with the replicated RNA segments assemble into new virus particles to be released from the host cell. RNA polymerases of avian influenza viruses function poorly in mammalian cells unless they undergo adaptive changes. Therefore the RNA polymerase can determine which hosts the virus infects (e.g. humans, birds or swine) and can also determine the severity and the outcome of the disease caused. However, we do not understand the molecular basis of these activities.
Our research programme aims to uncover how the RNA polymerase works and takes advantage of host co-factors in the infected cell. By understanding these processes we will uncover new ways of inhibiting the polymerase, and thus preventing the virus from multiplying, spreading and causing disease. We will also learn how genetic changes in the polymerase allow an influenza virus to jump from birds to humans and cause disease. Our bodies react to viral infection by producing substances that protect us from the virus. However, infection with 'bird flu' can cause our bodies to overreact by producing far too much of these substances, actually resulting in more severe disease. We found that the polymerases of 'bird flu' viruses do not work optimally in mammalian cells and produce aberrant short RNAs that we call mini viral RNAs (mvRNAs). These mvRNAs are very strong inducers of antiviral substances and could underlie the high virulence of H5N1 and H7N9 'bird flu' viruses in humans. By elucidating how these mvRNAs are made and what their effects are, we will be able to understand what makes 'bird flu' so deadly in humans. Working with industrial partners, this knowledge could also help with the design of improved influenza vaccines that are fine-tuned to induce a precise level of antiviral substances and to give the highest level of protection to those vaccinated.

Our research programme will uncover the role of the influenza virus RNA polymerase in virulence, host restriction and innate immune activation, and aid the development of novel antivirals and improved vaccines through investigating the structure-function relationships of the influenza virus transcriptional machinery. Specifically, we will determine how the transcriptase and replicase functions of the polymerase are regulated through interactions with host RNA polymerase II and dimerization. We will investigate the high-resolution structures of vRNPs and the replicative intermediate complementary RNPs (cRNPs) and will carry out a structural analysis of the vRNA in vRNPs. To identify accessible 'sensitive' sites in the polymerase that could be targeted by small molecule inhibitors, we will use a set of nanobodies that bind strongly to the polymerase and inhibit its function. We will investigate the role of the ANP32 family of host proteins, underlying polymerase mediated host restriction of avian influenza viruses in humans, and the mechanisms of influenza virus-induced host shut-off. To address how influenza viruses activate innate immune responses, we will investigate the molecular mechanisms by which the influenza virus polymerase generates mini viral RNAs (mvRNAs), that act as strong inducers of interferon expression and could underlie the high virulence of 1918 pandemic virus and avian influenza virus infections in humans.
To meet these objectives we will use an inter-disciplinary approach, combining methods such as x-ray crystallography, cryo-electron microscopy, SAXS, next generation sequencing, bioinformatics, bio-imaging, single-molecule analyses (smFRET), biochemical and virological methods, including reverse genetics for generating influenza virus mutants. The results will be fully exploited, in collaboration with industrial partners, towards developing antivirals targeting the viral RNA polymerase and the development of improved influenza vaccines.

The research programme tackles a serious threat to human health. Influenza viruses are a major contributor to disease and death in humans, and represent a considerable burden to healthcare systems globally. This research programme will use a basic science approach to better understand how the influenza virus transcribes and replicates its RNA genome and how this impacts the host range and virulence of influenza viruses. Through addressing these questions, we hope to identify novel features through which influenza virus infections could be controlled.
In the near term, the research programme will primarily impact the academic research sector. Presenting the high-resolution structures of human and avian influenza A virus polymerases and their various functional conformations will stimulate work by others into elucidating the mechanisms of these molecular machines. Influenza viruses belong to the group of negative strand RNA viruses, which include many human pathogens such as measles, rabies, respiratory syncytial virus, and Ebola. We expect that our work will also impact studies of the RNA polymerases of these other pathogens and stimulate the development of antivirals that target them. The next-generation sequencing data of RNAs from infected cells will represent a rich source of information on the interplay between influenza virus and the host cell, not only for the influenza research community but also for researchers working in the field of cellular transcription. Furthermore, the proposed research tackles the molecular basis of host range and virulence and will help with the assessment of the pandemic potential of emerging influenza virus strains. As such, it has the potential to impact policy-making, i.e. planning and executing an integrated response to influenza pandemics.
In the medium term, the identification of potential targets for antiviral drugs will stimulate research in academia and the pharmaceutical industry. This has the potential to lead to the development and licensing, in the long term, of novel anti-influenza drugs. Specifically, we aim to use our expertise and ability to generate large amounts of influenza virus polymerase for structural studies to screen for compounds with high binding affinity for the polymerase, in collaboration with industrial partners. Addressing how innate immune responses are triggered in influenza virus-infected cells and understanding the molecular mechanism behind the generation of mini viral RNAs (mvRNAs) and their role in triggering innate immune response could underpin efforts into the development of novel influenza vaccines. In turn, novel drugs and vaccines could contribute to the reduction of disease and human suffering, benefit health services through a reduction in bed residence and associated nursing and treatment costs, and the economy through reduced absence from work. In the case of an emerging pandemic, they might avert a global disaster.
The proposal will support the training of PDRAs employed on the programme, who will be working in an inter-disciplinary environment and will be exposed to the latest emerging technologies in structural analyses, single-molecule technologies, next generation sequencing, bioinformatics as well as virology. It will provide the PDRAs with training to either develop independent research of their own, or to transition to work effectively in industry.
Scientists employed on the proposed project will participate in public outreach activities contributing to increasing public awareness and understanding of the latest developments in medical research, for example by giving public talks and participating in the annual Oxford Science Festival. We will take every opportunity to communicate our research to schoolchildren to increase interest in natural sciences and foster interest, particularly from potential future scientists.