Replication of influenza virus at the molecular level

The fundamental aim of this proposed research programme is to develop a much deeper understanding of the molecular and cell biology of influenza viruses, allowing the development of novel strategies to combat influenza. Influenza viruses are a major contributor to disease and death in humans, and with their ability to cause yearly epidemics and occasional pandemics they represent a considerable burden to healthcare systems globally. The most devastating influenza pandemic, the 1918 "Spanish" flu, caused over 50 million deaths worldwide. Wild birds represent an important reservoir for influenza A viruses from which novel pandemic strains can emerge. Currently the H5N1, H7N7 and H9N2 subtypes are considered the biggest threats as these are prevalent in birds and can infect humans directly. Although these viruses lack the ability to transmit between humans, they can cause severe disease and death. The concern is that these viruses, through adaptation or by mixing with other virus subtypes, could gain the ability to easily transmit between humans and lead to a new pandemic. Although vaccines against seasonal influenza and a very limited number of antiviral drugs are available, we are surprisingly defenceless against emerging pandemic influenza viruses. To prepare a vaccine matching a pandemic strain could take several months and resistance to drugs is an increasing problem. Therefore novel strategies for the control of influenza need to be developed.
The focus of the proposed research programme is the transcriptional machinery of influenza virus, a complex of several proteins which is responsible for copying the genetic information of the virus in a host cell. The genetic information of influenza virus is stored in segments of RNA. These need to be copied into messenger RNA (transcription) to be translated into viral proteins, as well as replicated to produce new copies of the genome. These new copies are then assembled with viral proteins to be incorporated into new virus particles that are released from the host cell to initiate new cycles of viral reproduction. Interestingly, transcriptional machineries of influenza viruses that infect birds tend to work poorly in human cells. When "bird flu" transmits to humans the machinery usually undergoes changes to become adapted to its new host. In other words, the transcriptional machinery of an influenza virus can determine which host the virus could infect, and can also determine the severity and the outcome of the disease caused.
Although the viral transcriptional machinery performs essential functions in a host cell-specific manner without which the virus cannot survive, we do not understand how this machine works at the molecular level or how the host cell's species (whether bird or human) affects its function. We therefore wish to determine the molecular mechanisms used by the viral transcriptional machinery to transcribe and replicate the viral genome, and the molecular interactions between the viral transcriptional machinery and the host cell. This research programme will help to understand the molecular basis of virulence, host range restriction and adaptation of influenza viruses to new hosts. It will therefore allow a better understanding of the emergence of strains with pandemic potential. Importantly, it will underpin the development of novel antiviral strategies by indentifying targets for antiviral drugs in the viral transcriptional machinery as well as in cellular proteins. It could also facilitate the development of novel influenza vaccines, offering protection against a wide variety of influenza virus strains. Any emerging information during the course of the programme will be exploited, potentially with the involvement of industrial partners.

The proposed research programme aims to uncover fundamental aspects of structure and function of influenza virus as well as virus-host interactions and host responses to viral infection. The main focus is the viral transcriptional machinery, a multi-subunit enzyme complex, which transcribes and replicates the viral RNA genome in a host cell-dependent manner. This enzyme, which consists of the viral RNA polymerase and nucleoprotein, is essential for the virus to replicate and is an important determinant of host range and virulence. The research objectives are to determine the molecular mechanisms of transcription and replication of the viral RNA genome by this transcriptional machinery, to identify and characterise the molecular interactions it makes with the host cell, and to determine the implications of these interactions for cell tropism, host range and virulence. The proposal builds on the achievements of my research over a 20 year period, in the last 10 years supported by a Senior Research Fellowship and 5-year Research Grant from the MRC. We will use the latest state-of-the-art technologies in single-molecule imaging (FRET), super-resolution microscopy (PALM and STORM), structural biology and proteomics in combination with methods from biochemistry, cell biology and virology. Recombinant proteins for structural analysis will be expressed using recombinant baculovirus technology (Multi-Bac) allowing the expression of multiple proteins from a single baculovirus in insect cells. Influenza virus mutants for cell biology and animal studies will be generated by reverse genetics. Virulence studies will use established animal models for influenza. Impacts of this research will include understanding the emergence of influenza strains with pandemic potential, discovery of novel viral and cellular drug targets, and the development of novel vaccines by reverse genetics. Promising avenues will be exploited in collaboration with industrial partners.

The proposed research programme focuses on fundamental aspects of structure and function of influenza viruses as well as on virus-host interactions and host responses to viral infection. Influenza viruses are a major contributor to disease and death in humans, and with their ability to cause yearly epidemics and occasional pandemics they represent a considerable burden to healthcare systems globally. It is estimated that influenza is responsible for between 3 million and 5 million cases of severe illness and between 250,000 and 500,000 deaths worldwide annually. Influenza could be considered one of today's biggest threats to the world's socio-economic health, and the threat of a novel pandemic influenza virus emerging in the human population against which there would be no pre-existing immunity remains a cause for serious concern. The UK National Security Council in its Strategic Defence Review (published on 18 October 2010) classified a potential influenza pandemic in the group of risks of the highest priority for UK national security.
In the shorter term (year 2-3 of the programme), the research programme will uncover molecular mechanisms used by influenza viruses to adapt to a new host and it will therefore be useful in identifying influenza viruses with the potential to cause a pandemic. This knowledge could contribute towards evidence-based policy making and influence public policies on influenza pandemic preparedness.
In the longer term (beyond 5 years), the research programme has the potential to impact the public by underpinning the development of antiviral drugs and improved vaccines. 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, and in the case of an emerging pandemic they might avert a global disaster. This long-term aim will be fully exploited with the involvement of new collaborators, specialists in structure-based drug design and vaccine development as well as industrial partners depending on the outcomes of research.
Any outcomes with potential of being commercially exploited will be communicated to Isis Innovation Limited, a wholly-owned subsidiary of the University of Oxford, managing technology transfer and academic consulting for scientists working at the University of Oxford. The project also has the potential to contribute to wealth creation and economic prosperity through the creation and growth of companies and jobs, enhancing business revenue and innovative capacity. This could be achieved by the commercialisation and exploitation of scientific knowledge, leading to spin-off companies and the creation of new drugs and vaccines.
The project will also contribute to training and delivering highly skilled researchers. PDRAs employed on the project will gain skills in virology as well as in novel emerging technologies (e.g. single-molecule technologies, super-resolution microscopy, proteomics) that will allow them to take leading positions in various fields of industry, e.g. in drug and vaccine development not only against influenza virus, but also against other significant human and animal pathogens.
Scientists employed on the project will participate in public outreach activities contributing to increasing public awareness and understanding of the latest developments in influenza virus 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.