Utilizing the fusion machinery of Herpes Simplex Virus to unveil the general process of membrane fusion

Project Information

KGBVIFEF

Grant agreement ID: 654154

DOI

10.3030/654154

Project closed

EC signature date 25 March 2015

Start date 1 May 2015

End date 30 April 2017

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 454,80

EU contribution

€ 183 454,80

Coordinated by

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom

Periodic Reporting for period 1 - KGBVIFEF (Utilizing the fusion machinery of Herpes Simplex Virus to unveil the general process of membrane fusion)

Reporting period: 2015-05-01 to 2017-04-30

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

As cryoEM was a central element of the project, emphasis was given to training in its state-of-the-art aspects e.g. via workshops, training sessions and seminars and now allows me to confidently use and troubleshoot latest technology microscopes and accessories plus the dedicated software. For image analysis and processing I participated in group and university internal training as well as an EMBO workshop now allowing me to use a number of programs for image processing, tomogram reconstruction, subvolume averaging and single particle 3D reconstruction.
My initial familiarisation with the topic and necessary methods was followed by tests to purify and reconstitute proteins to establish the core fusion machinery in vitro to investigate the fusion process. Overexpression of the full-length, main fusion protein gB was found to lead to formation of large intracellular membrane vesicles, accompanied by release of membrane protein enriched extracellular vesicles (MPEEVs). On these MPEEVs a conformation distinct from the previously known post-fusion structure was observed. Immunogold labelling and mass spectrometry identified this structure as gB. CryoET in combination with sub-volume averaging was used to solve the first full-length structure of a class III fusion protein in a pre-fusion state. The results of this study are published in (Zeev-Ben-Mordehai et al. PNAS 2016).
Investigation and optimisation of the system now allows purification of high concentrations of gB MPEEVs which are used in an approach to collect more cryoEM data sets using latest technology to achieve higher resolution structures. One approach uses cryoET of MPEEVs and sub-tomogram averaging, the other uses the full-length protein, solubilised and reconstituted in lipid discs for single particle analysis. Resulting structures will be made available on http://www.emdatabank.org.
Apart from gB, other members of the complex - gH/L or gD - or combinations thereof were not found in MPEEVs. Fluorescent microscopic investigations revealed that interaction of the fusion complex members is either transient during fusion or is mediated via additional factors. The large intracellular vesicles, caused by expression of gB were identified as multivesicular bodies, part of the endosomal pathway. These findings shed light on gB trafficking, viral assembly and formation of extracellular vesicles, which in recent years came into broader interest as valuable biomarkers.
To efficiently identify the fusion trigger and to reconstitute and analyse the fusion event in vitro, an experimental system featuring giant unilamellar vesicles was designed and set up. This system allows reproducible production of vesicles of defined lipid compositions and reconstitution of transmembrane proteins for light microscopic evaluation of the fusion process. Artificial membrane tethering of soluble fragments of gD and gH/L was successful and will be used as minimal system to reconstitute the fusion process.
Results obtained during the project were shared and discussed on scientific conferences and workshops. The main findings so far were published in Zeev-Ben-Mordehai et al. PNAS 2016 and further publications are in preparation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

About 26% of the proteins encoded in the human genome are predicted to contain at least one transmembrane domain (Fagerberg et al. Proteomics 2010). However, of all solved structures in the Protein Data Bank (www.rcsb.org) only ~2.5% are membrane proteins.
Expression and purification of these proteins is difficult and requires detergent which often compromises structural integrity. Hence, alternative methods are highly sought after. My project focused on a complex including three transmembrane proteins. A novel approach to reconstitute proteins after solubilisation into membrane discs was tested using MPEEVs as expression platform to obtain full length proteins as starting material and the encouraging results show the feasibility of this approach. Although too low for crystallisation, achievable concentrations with this system are sufficient for characterisation by cryoEM and biochemical assays. Combination of vesicle expression with nanodiscs and single particle cryoEM is an attractive workflow that would allow the structural characterisation of a wide range of yet uncharacterised transmembrane proteins, including receptors and transporters that are potential drug targets. To test the general applicability, internal and external collaborations were actively initiated and the underlying mechanism was further investigated in parallel. The outcomes will be used to optimise the approach to make it wider applicable and to expand to interaction studies between membrane proteins.

Method for structure determination of Glycoprotein B

After gD interacts with cell surface receptors gH/L is initiated to activate gB for membrane fusion.