Molecular mechanisms of M2-mediated influenza virus budding and scission

Influenza virus is a pathogen of great medical importance that has dominated the headlines many times in recent years. While seasonal epidemics of influenza exact significant socio-economic costs it is the emergence of new and novel strains of influenza virus that provide the most cause for concern. In 2009 the newly-emerged 'swine flu' strain of influenza virus quickly spread around the world causing a pandemic that taxed heath care systems throughout the world. Since 2003, fears over 'avian influenza' have lead to the culling of livestock and have significantly impacted the tourism industry in Asia. While avian influenza viruses are not as contagious for humans as swine-origin influenza viruses they are much more pathogenic, causing fatalities in 60 % of the cases. There is a significant fear that mutation of avian influenza viruses will occur, allowing them to spread from human-to-human with the efficiency of swine strains while retaining their high rates of mortality. This is highlighted by the recent emergence of H7N9 avian influenza viruses in China that have caused 130 cases of human infection in April 2013. The result of an emergent transmissible strain could be similar to that which occurred in 1918 when an influenza virus pandemic caused the death of 1 % of the world's population from 1918-1918. Thus, there is a continued need for the greater understanding of this deadly virus in the hopes of generating new methods to control and treat influenza virus infections.

Influenza virus is an enveloped virus that obtains its lipid shell from the host cell's plasma membrane through a budding process. Inserted in the virus envelope are three proteins: hemagglutinin (which mediated viral entry into the host cell), neuraminidase (which is essential for the release of budded viruses) and the M2 protein (which is an ion channel protein that plays an important role during virus entry). Among the least understood events of the influenza virus lifecycle are the processes that mediate the formation of new viruses and their release from the host cell by membrane fission. Recently we have found a novel role for the influenza virus M2 protein in virus budding, the mediation of membrane fission. The aim of this research is to investigate the function of the influenza virus M2 protein to better understand its involvement in virus budding, allowing for a greater understanding of the complex process of influenza virus assembly and budding.

The completion of this research project will show how the M2 protein is able to facilitate the budding of influenza viruses. However, this research has greater implications beyond the understanding of M2 protein functions. First, the creation of in vitro systems for investigating virus budding will be a valuable research tool allowing for other scientists to investigate the assembly and budding of multiple different viruses and protein complexes. Secondly, detailed understanding of the mechanism by which the M2 protein accomplishes its essential task of membrane fission will allow for the development of anti-influenza drugs targeting this function. Finally, better understanding of how the complex processes of virus assembly and budding are accomplished will advance the influenza virus field, generating essential knowledge about this medically important pathogen and providing many other new targets for anti-viral drug development.

Recently we have found an essential role for the influenza virus M2 protein amphipathic helix (AH) in the mediation of membrane scission and the release of budding viruses. This research will elucidate the molecular mechanisms of M2-mediated influenza virus budding and has the following objectives:

1. To determine the molecular mechanism by which the M2 AH alters membrane curvature;
2. To correlate M2-mediated alterations in membrane curvature with the process of membrane scission in vivo;
3. To determine the biophysical properties of M2-mediated membrane scission.

Each of these objectives will be addressed using a novel collection of state-of-the-art experimental protocols. To elucidate the mechanism by which the M2 AH alters membrane curvature, strategic mutations will be made in M2 peptides and their capacity for budding assessed in Giant Unilamellar Vesicles (GUV) containing multiple different lipid mixtures. The conditions and mutations that significantly affect M2-mediated budding will then be tested in vivo using a novel method for quantifying membrane budding in cellular membranes and by incorporation of the relevant mutations into recombinant viruses. These mutant viruses will then be assessed for function through a variety of assays including: viral assays (growth rate and budding efficiency) and protein assays (cholesterol association, protein incorporation and localization). These functional studies will be supported by biophysical studies of the M2 AH using real-time single GUV imaging and molecular dynamics simulations.

The successful completion of this research project will determine the molecular mechanism of M2-mediated budding. This research has significant implications in the understanding of influenza virus budding, establishes in vitro model systems that can be used in the investigation of budding for multiple different viruses, and provides the molecular details necessary for future drug discovery research.

While this research has direct primary benefit to the academic research sector it also has two main benefits outside of the academic research environment. The first benefit pertains to the potential of pharmaceutical development following the completion of these studies. The elucidation of the molecular mechanisms behind influenza virus assembly and budding will offer multiple different candidates for the rational design of novel therapeutic agents. As such, biotechnology companies in the commercial private sector are well positioned to take advantage of the results of this research. The exploitation of these research results by the biotechnology sector also provides an added beneficiary of this research as there is now potential development of new therapeutic agents to treat and prevent influenza. This development would have significant benefits to public health, could affect public health policy for the treatment and prevention of influenza and would be of benefit to the UK economy (both for the economic advantage of the development of a highly marketable pharmaceutical as well as the alleviation of the socio-economic costs of influenza disease and treatment).

This research will also be of benefit to the public. Influenza is a disease that is found in newspaper headlines around the world. The terms 'swine flu', 'avian influenza' and 'the great pandemic' cause great fear in many countries around the world and there is a significant public demand for new treatments and preventative measures. The successful completion of this research project will provide new understandings of influenza virus budding and new targets for drug development. The knowledge that progress is being made towards the development of new therapeutics for influenza treatment and prevention will be a crucial measure in alleviating public fear and providing reassurance that significant steps are being taken to prevent and combat the influenza virus.