Molecular mechanisms of M2-mediated influenza virus budding and scission

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Badham MD (2016) Filamentous Influenza Viruses. in Current clinical microbiology reports

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Madsen JJ (2018) Entropic forces drive clustering and spatial localization of influenza A M2 during viral budding. in Proceedings of the National Academy of Sciences of the United States of America

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Martyna A (2014) Alterations of membrane curvature during influenza virus budding. in Biochemical Society transactions

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Martyna A (2016) Curvature Sensing by a Viral Scission Protein. in Biochemistry

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Michaelis M (2016) Computational analysis of Ebolavirus data: prospects, promises and challenges. in Biochemical Society transactions

 
Description Business Interaction Voucher
Amount £10,000 (GBP)
Funding ID D0104 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 12/2016
 
Description Investigator Award
Amount £490,000 (GBP)
Funding ID MR/L018578/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2014 
End 03/2017
 
Description Medical Research Council CASE Studentship
Amount £95,327 (GBP)
Funding ID MR/L015560/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 08/2018
 
Description Collaboration with D.I.Ivanovsky Institute of Virology 
Organisation Russian Academy of Medical Sciences
Department Ivanovsky Institute of Virology Russian Academy of Medical Sciences (IVIV)
Country Russian Federation 
Sector Hospitals 
PI Contribution Analysis of viral morphology, manuscript content editing.
Collaborator Contribution Key research on viral morpholgy and the entry of influenza viruses.
Impact Zhirnov O., Manykin A., Rossman J.S., Klenk H. (2016) Intravirion cohesion of matrix M1 with ribonucleocapsid is a prerequisite of Influenza virus infectivity. Virology. In Press. Research grant application: International Exchanges Scheme - 2016 RFBR Russian Cost share.
Start Year 2016
 
Description Collaboration with Mattek 
Organisation MatTek Corporation
Country United States 
Sector Private 
PI Contribution We are utilising Mattek's 3D differentiated airway tissue culture systems in order to investigate influenza virus assembly and budding.
Collaborator Contribution Mattek has provided tools, reagents and expertise towards this project.
Impact Biotechnology and Biosciences Research Council, Crossing Biological Membranes, Business Interaction Award with MatTek (D0104), Optimization of influenza vaccine manufacturing through modification of cellular membrane organization, September-December 2016, £10,000. Completed.
Start Year 2016
 
Description Collaboration with MedImmune 
Organisation MedImmune Ltd
Country United Kingdom 
Sector Private 
PI Contribution Investigation of influenza virus assembly and budding as pertaining to the filamentous morphology of influenza viruses, with the aims of altering morphology to increase vaccine production.
Collaborator Contribution Financial contributions, contributions of reagents and resources, contributions of expertise, training and access to facilities.
Impact Medical Research Council CASE Studentship with MedImmune LLC (MR/L015560/1), Optimization of influenza vaccine development by modification of viral morphology, September 2014-August 2018, £ 95,327.
Start Year 2014
 
Description Collaboration with Oxford 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative research investigating the role of M1 and M2 proteins in influenza virus assembly and budding.
Collaborator Contribution Collaborative research investigating the role of M1 and M2 proteins in influenza virus assembly and budding.
Impact Medical Research Council Research Grant (MR/L018578/1), Cytoplasmic tail interactions of the influenza M2 protein with lipid and protein, Co-I with the University of Oxford, April 2014-March 2017, £490,000.
Start Year 2013
 
Description Collaboration with Stockholm University 
Organisation Stockholm University
Country Sweden 
Sector Academic/University 
PI Contribution Research into the role of the influenza virus NA protein during virus assembly and budding.
Collaborator Contribution Research into the role of the influenza virus NA protein during virus assembly and budding.
Impact da Silva, D., Nordholm, J., Dou, D., Wang, H., Rossman, J.S. and Daniels, R. (2014) The influenza NA protein transmembrane and head domains have co-evolved J. Virol. 89(2): 1094-104.
Start Year 2014
 
Description Collaboration with the Ohio State University 
Organisation Ohio State University
Department Child and Adolescent Psychiatry
Country United States 
Sector Hospitals 
PI Contribution Research collaboration investigating the mechanisms of budding/entry of cell penetrating peptides, active collaboration by both partners.
Collaborator Contribution Research collaboration investigating the mechanisms of budding/entry of cell penetrating peptides, active collaboration by both partners.
Impact Qian, Z, Martyna, A., Hard, R., Wang, J., Appiah-Kubi, G., Coss, C., Phelps, M., Rossman, J.S. and Pei, D. (2016) Discovery and mechanism of highly efficient cyclic cell-penetrating peptides. ACS Biochemistry 55(18): 2601-2612.
Start Year 2016
 
Description Cell Biology conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Royal Society Meeting on Cell adhesion, London
Year(s) Of Engagement Activity 2014
 
Description Media Interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Served as a virology expert during the 2014-2015 Ebola virus outbreak and the on-going Zika virus outbreak, participating in several interviews (Sky TV, ITV, BBC Radio Kent, The Conversation, KMFM Radio).
Year(s) Of Engagement Activity 2015,2016,2017
 
Description Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Seminar on influenza virus budding to The Pirbright Insitute, Compton, UK,
Year(s) Of Engagement Activity 2015
 
Description University Open Day Seminar 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Science talk on influenza and virology during the University of Kent Open Days 2016-2017. These events generated significant interest with both parents and students.
Year(s) Of Engagement Activity 2016,2017
 
Description Virology Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at the Options IX for the Control of Influenza meeting in Chicago, IL, USA.
Year(s) Of Engagement Activity 2016
 
Description Virology conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 4-6th International Influenza Meetings, University of Muenster, Germany
Year(s) Of Engagement Activity 2014,2015,2016