Final Report Summary - INFLUENZA BUDDING (Mechanisms of Influenza virus assembly and budding)
Influenza virus is a medically-important pathogen with significant socio-economic costs and is a leading cause of morbidity and mortality world-wide. There remains a need for the effective control of influenza viruses by a new generation of therapeutic agents. This research is designed to investigate the assembly and budding of influenza viruses with the hope that the knowledge gained though these studies will provide new targets for anti-influenza drug development.
The objectives of the four-year project were: to determine the structure of the M2 cytoplasmic tail, elucidate its essential functions during influenza virus budding and to determine the molecular mechanism by which the M2 amphipathic helix alters membrane curvature and causes membrane scission and finally to reconstitute influenza virus budding in vitro.
Over the course of this project we have made extensive progress in determining the role of the influenza virus M2 protein in virus budding. We have solved the structure of the M2 amphipathic helix bound to membrane, a result that provides essential information on how the M2 protein alters membrane curvature and mediates scission during influenza virus budding. Furthermore, we have completed a panel of biophysical and virological experiments designed to identify key functional regions of the M2 cytoplasmic tail and evaluate their impact on virus budding. From these studies we have determined that M2 has a novel membrane localization during virus budding that is dependent on the polar face of the M2 amphipathic helix and that plays an essential role in virus assembly and budding. These results have allowed us to attempt the reconstitution of influenza virus budding in vitro. Our results have shown that a collection of viral proteins is required for the assembly and release of virus-like particles that resemble influenza virions. We see that HA, NA, M2 and M1 proteins all play crucial roles in viral structuring, and that each protein makes defined contacts with specific lipid environments and other viral proteins. M2 and HA interact though lipid domain association whilst the NA transmembrane domain is a key determinant for the initiation of virus assembly and budding. However, attempts to work with soluble M1 protein have failed and thus, complete in vitro assembly has not been possible. Current attempts are ongoing and will form the basis of a future research grant application.
Results from project have directly led to six research articles with a seventh currently in preparation. Project support has also enabled preliminary experiments that formed the basis of a further eight published articles. In addition, data generated from this project has resulted in the awarding of a UK Medical Research Council Investigator Grant, greatly enhancing the PI’s career integration into the European research community. The results of this project have significantly advanced the field of virus budding beyond the current state of the art. Previously it was unknown how membrane scission occurs during virus budding. This is of exceptional interest, as identifying the molecular details of virus assembly can offer multiple potential targets for antiviral therapeutic development. Our results clearly define the molecular mechanism of scission that is necessary for the release of all influenza viruses. These results identify key protein-protein and protein-lipid interactions that are targets for potential therapeutics and we are already designing screens and modelling programs that will identify novel anti-influenza compounds. Thus, these results not only further our understanding of influenza virus budding, but also have a significant public impact as it may be possible to create a new generation of therapeutics to combat the serious public health threat that is influenza virus.
However, the impact of our results is not limited to the field of virus budding. Our results from have already provided mechanistic understanding of the process of amphipathic helix-driven membrane scission. Many different cellular proteins possess amphipathic helices and have been implicated in various cellular budding events. Our data suggest that there may be a family of (virus and cellular) AH-containing proteins that all mediate membrane scission by similar mechanisms. Thus, the M2 AH may serve as a model scission helix and provide better understanding of other complex budding events, such as clathrin-independent endocytosis and multi-vesicular body budding.
Dr Jeremy Rossman
School of Biosciences
University of Kent
Canterbury, UK
http://www.kent.ac.uk/bio/profiles/staff/rossman.html
The objectives of the four-year project were: to determine the structure of the M2 cytoplasmic tail, elucidate its essential functions during influenza virus budding and to determine the molecular mechanism by which the M2 amphipathic helix alters membrane curvature and causes membrane scission and finally to reconstitute influenza virus budding in vitro.
Over the course of this project we have made extensive progress in determining the role of the influenza virus M2 protein in virus budding. We have solved the structure of the M2 amphipathic helix bound to membrane, a result that provides essential information on how the M2 protein alters membrane curvature and mediates scission during influenza virus budding. Furthermore, we have completed a panel of biophysical and virological experiments designed to identify key functional regions of the M2 cytoplasmic tail and evaluate their impact on virus budding. From these studies we have determined that M2 has a novel membrane localization during virus budding that is dependent on the polar face of the M2 amphipathic helix and that plays an essential role in virus assembly and budding. These results have allowed us to attempt the reconstitution of influenza virus budding in vitro. Our results have shown that a collection of viral proteins is required for the assembly and release of virus-like particles that resemble influenza virions. We see that HA, NA, M2 and M1 proteins all play crucial roles in viral structuring, and that each protein makes defined contacts with specific lipid environments and other viral proteins. M2 and HA interact though lipid domain association whilst the NA transmembrane domain is a key determinant for the initiation of virus assembly and budding. However, attempts to work with soluble M1 protein have failed and thus, complete in vitro assembly has not been possible. Current attempts are ongoing and will form the basis of a future research grant application.
Results from project have directly led to six research articles with a seventh currently in preparation. Project support has also enabled preliminary experiments that formed the basis of a further eight published articles. In addition, data generated from this project has resulted in the awarding of a UK Medical Research Council Investigator Grant, greatly enhancing the PI’s career integration into the European research community. The results of this project have significantly advanced the field of virus budding beyond the current state of the art. Previously it was unknown how membrane scission occurs during virus budding. This is of exceptional interest, as identifying the molecular details of virus assembly can offer multiple potential targets for antiviral therapeutic development. Our results clearly define the molecular mechanism of scission that is necessary for the release of all influenza viruses. These results identify key protein-protein and protein-lipid interactions that are targets for potential therapeutics and we are already designing screens and modelling programs that will identify novel anti-influenza compounds. Thus, these results not only further our understanding of influenza virus budding, but also have a significant public impact as it may be possible to create a new generation of therapeutics to combat the serious public health threat that is influenza virus.
However, the impact of our results is not limited to the field of virus budding. Our results from have already provided mechanistic understanding of the process of amphipathic helix-driven membrane scission. Many different cellular proteins possess amphipathic helices and have been implicated in various cellular budding events. Our data suggest that there may be a family of (virus and cellular) AH-containing proteins that all mediate membrane scission by similar mechanisms. Thus, the M2 AH may serve as a model scission helix and provide better understanding of other complex budding events, such as clathrin-independent endocytosis and multi-vesicular body budding.
Dr Jeremy Rossman
School of Biosciences
University of Kent
Canterbury, UK
http://www.kent.ac.uk/bio/profiles/staff/rossman.html