Cytoplasmic tail interactions of the influenza M2 protein with lipid and protein.

Influenza is an easily spreadable acute infection of the influenza virus that can result in severe illness and death, particularly for young children and older adults. Globally, 3-5 million people suffer severe illness and 250,000-500,000 people die from influenza every year (World Health Organization estimates). Prevention of Influenza through vaccination remains the most effective control. However, the required lead times for vaccine production and the fast evolution of circulating viruses require that development of the vaccines are based on predictions for future circulating strains. As a result, vaccine effectiveness varies widely year-to-year. Although the effectiveness rates are sometimes as high as 90%, the 2012 vaccine effectiveness was below 50%. In addition, drugs may be developed that can prevent an influenza virus infection or limit the seriousness of an infection that has occurred. Drugs that target the viral neuraminidase have been developed (oseltamivir and zanamivir). However, the efficacy of these drugs is variable and resistance to these drugs for some influenza strains is common and for others is increasing. A much older class of antivirals, the amino-adamantane (AA) drugs (rimantadine and amantadine) target the viral M2 protein and have been used for decades to treat influenza in humans and livestock. However the last decade has seen a dramatic increase in resistance to this class of drugs. The vast majority of the seasonal strains including the 2009 H1N1 "swine flu" pandemic and the 2013 H7N9 outbreak in China were resistant to the AA drugs, with swine flu M2 being particularly insensitive. However, the AA drugs, target a single activity of the M2 protein - ion channel activity. M2 is now known to possess additional activities related to the formation of new virus particles, and these activities are attributable to regions of the protein not associated with the ion channel activity. These activities include induction of host cell membrane deformation as well as specific interactions with other viral proteins that facilitate assembly of new virus particles. Our work aims to characterise these interactions at the molecular level that will enable refinement and extension of current models of the viral life cycle. These studies may ultimately pinpoint promising therapeutic approaches to treating influenza.

The aim of this grant is to provide structural and functional information on the cytoplasmic tail of the influenza virus protein M2 and its interactions with the membrane, with specific lipids, and with M1. The amino-adamantane drugs, which target the ion channel activity of M2, have been used as antivirals for decades, but currently circulating strains are resistant. However, M2 has recently discovered functions in the budding and scission of nascent viral particles. A membrane proximal amphipathic helix (APH) is implicated in generating the negative membrane curvature required for demarcating the viral budozone. In addition, M2 interacts with the viral protein M1, which facilitates organisation of nascent virion. The cytoplasmic C-terminal domain of M2 is responsible for both membrane and M1 interactions. Whereas the membrane proximal APH has been characterised in some detail by NMR and EPR methods, less is known about the high resolution details of the depth of insertion of the APH in the membrane and the specific interactions with lipids, including cholesterol and PIP2. For the M2/M1 interactions, the M2 residues involved have been mapped to the region around residues ~71-76. However, nothing is known about the corresponding site on M1 and whether M1 or M2 undergoes any conformational changes upon binding. In addition, there are no experimental reports of the site-specific structural elements, if any, in the M2 cytoplasmic tail outside of the APH (residues ~63-97). Therefore, we aim to characterise M2 interactions with the membrane, in particular that of the APH, and correlate these findings across variants and cholesterol concentration with the activity of M2 in GUV membrane budding assays. In addition, we will characterise structurally and functionally the interactions of the M2 APH with both cholesterol and PIP2. Finally, we will determine the molecular basis of M2/M1 interactions and whether complex formation leads to structural rearrangements.

The general areas of impact are to: (i) increase biophysical and biological understanding of influenza virus budding and scission, (ii) identify the molecular basis of a protein-protein and protein-lipid interactions involved in organising budding influenza virions with the potential to influence therapeutic target development, (iii) significantly increase the molecular understanding of protein-lipid interactions, and (iv) provide training in the study of structure-function correlations in membrane proteins using biophysical, biochemical and biological methods.
The impact of the proposed research will extend beyond the M2 system that is to be studied, since a large number of membrane proteins have been identified as important in membrane deformation but few of their membrane interactions have been characterised in atomic detail. As an example of a membrane-deforming protein, interactions of M2 with the membrane and specific lipids will help develop our general understanding of protein interactions with membranes.
The primary mechanism for communication of this research will be through publication in peer review international journals. Open access publishing options will be used where available. We will liaise at the time of publication with the University of Oxford, the University of Kent, and MRC press offices to ensure wide dissemination of results that are of interest to the general public. We will also take advantage of opportunities to communicate via freely accessible media (such as the University of Oxford Department of Biochemistry and University of Kent School of Biosciences websites) in order to extend the impact of the findings. Our results will also be made available on our regularly updated laboratory web sites. Data obtained during this project, such as structural models and NMR chemical shift assignments will be deposited into the open-access Protein Data Bank and BioMagResBank databases, respectively.
PDRA Claridge employed on this grant will gain technical skills in manipulation of protein and lipid samples and their characterisation using the latest advances in NMR spectroscopy and biophysical methods. Most notably, he will become an expert in the physicochemical properties of bicelles and lipid nanodiscs, which are rapidly becoming important tools for studying membrane proteins in near-native conditions. In addition he will be trained in writing, IT, and presentational skills, and will benefit from working closely with expert colleagues using different but complementary techniques, thereby enhancing his future research employment prospects.