Membrane fusion is a basic mechanisms of cell biology, but its molecular details remain poorly understood. A better knowledge will advance the understanding of a multitude of processes ranging from initial steps of pathogenic attack to developmental diseases and help developing new therapeutic drugs.
Cell entry of Herpes simplex virus-1 (HSV-1) by membrane fusion is supposedly mediated by the surface glycoprotein gB. Unlike many other viral fusion proteins, the process including the initial recognition, approximation and fusion of the two membranes, plus its regulation is distributed to four glycoproteins, namely gB, gD and gH/gL.
The overall objective of this project was to take advantage of this modularity to better understand the individual steps of the fusion process and hence HSV-1 cell entry.
This approach included the structural determination of the full-length, membrane bound fusogen gB as well as investigating its interaction with other fusion complex members. By reconstitution of the protein complex in vitro and determination of the fusion trigger, the cascade of events during fusion was to be analysed in molecular detail. This information would then be applied to determine the structural arrangement of the full complex on the virus particle.
The project employed a multidisciplinary, structure-functional study combining different methods and data from structural biology, biochemistry and biophysics. This includes fluorescence microscopy, full-length membrane glycoprotein purification and biochemical reconstitution, but also in-depth training in and application of state-of-the-art electron cryo microscopy/tomography (cryo-EM/ET), sub-volume averaging, classification and single particle imaging.
In the course of this project significant advances have been made towards understanding HSV-1 membrane fusion. This includes structural determination of the full-length, membrane anchored fusion protein gB in two distinct conformations (one is novel) and its processing and interaction with other fusion complex members. As complex formation requires a deeper understanding of the regulation of interaction, an expansion of the approach, including further technologies was necessary and deciphering of the full mechanism is still ongoing.
Integral to the project a full personal training in latest, high resolution imaging technology and qualifying me now as highly skilled scientist in state-of-the-art cryoEM and also allowed me to set up an extensive scientific network at conferences, workshops and via numerous collaborations, helping me to establish myself as independent scientist in the field of European life science.
