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Discerning the mechanisms of cell membrane budding by coronavirus non structured proteins nsp3, nsp4 and nsp6


Project Description

Coronaviruses are positive strand- non-segmented RNA viruses, infect a wide range of hosts including mammalian species and have recently caused the severe acute respiratory syndrome (SARS), the Middle-East respiratory syndrome (MERS) and the current SARS CoV-2 (Covid-19) pandemic. In all these cases, the original reservoir of the coronavirus is thought to be bats, from which it crossed to humans via an intermediate host. Given the large diversity of coronaviruses in bats (estimated 1-6 ꞏ103 different coronaviruses), it is very likely that more cases of human infection by new coronaviruses will occur and a better knowledge of the principle mechanisms of virus infection and replication within host cells would be crucial for the development of new treatment strategies.

After the entry of the virus into the host cell, a key set of proteins, the non-structural proteins (nsps) nsp3, nsp4 and nsp6 are central in deforming the membrane of the endoplasmatic reticulum into double membrane vesicles (DMVs) together with membrane proteins of the host cell (Hagemeijer et al., 2014). These DMVs then serve as a platform for the viral replication and transcription complexes. Assembly of the DMVs is important for successful viral replication as they ensure optimal spatial organisation of the cellular and viral constituents required for RNA synthesis and segregation of the generated viral RNA species from the host defence mechanisms. Despite the conservation of the nsp-driven DMV formation among the coronaviruses, little is known about the biophysical and biochemical fundamentals of this process.

The aim of the PhD project is to study the membrane deformation and DMV formation induced by the proteins nsp3, nsp4 and nsp6 from two viruses, SARS coronavirus and SARS-Cov-2, using a minimal, reconstituted system. Giant unilamellar vesicles (GUVs) are a well-established model system, to study membrane protein interactions on cell sized lipid membrane vesicles with a defined lipid composition and readily visualised by optical fluorescence microscopy. Addition of varying combinations of purified full-length, fragments or peptides of nsp3, nsp4 and nsp6 to GUVs will allow us to identify the role of lipid composition and protein interactions in membrane binding and membrane deformation (Alsaadi et al., 2019). Biophysical measurements of the GUVs such as micropipette aspiration and optical tweezers assisted membrane tether extraction will allow us to study the role of physical parameters, such as membrane tension or bending rigidity, in nsp-driven membrane budding. Lipid-selective binding of the nsps will be evaluated using fluorescence spectroscopy measurements of Trp residues in the sequence. Circular dichroism and chemical cross-linking / SDS-PAGE will be used to evaluate protein folding and interactions, respectively, in GUVs of varying size and curvature (as evaluated using dynamic light scattering) (Brooks and Dixon, 2020).

Objectives:

- Reconstitution of membrane budding by a combination of purified nsp3, nsp4 and nsp6;
- Identification of the role of lipid composition in nsps binding and membrane deformation;
- Detection of changes in biophysical properties of nsps upon binding to GUVs;
- Study of the interaction dynamics of nsps on lipid membranes and the resulting membrane deformations.

The PhD project would be conducted in close collaboration between the Dixon and the Koester labs. The Koester lab is located at the Centre for Mechanochemical Cell Biology and works on the biophysics of lipid membrane-based systems. The lab has experience in the reconstitution of biomimetic protein-lipid membrane systems, GUV formation, micropipette aspiration, live fluorescence microscopy as well as fluorescence life-time microscopy. The in-house lattice light sheet microscope would be used to image nsp-induced membrane deformations with high spatiotemporal resolution. The Dixon lab is located in the Department of Chemistry and is focussed on work in the areas of membrane protein folding and protein-protein/protein-lipid interactions using a wide variety of biophysical and biochemical methods. The lab is well-equipped with all of the instrumentation described in the project outline. Dixon’s laboratory has a strong track record in the elucidation of sequence/structure relationships for TM protein and protein-lipid interactions, as well as characterisation of model membrane systems.

Techniques that will be undertaken during the project:

- Fluorescence microscopy: total internal reflection fluorescence (TIRF), confocal, lattice-light-sheet (LLS) and super-resolution (e.g. STORM)
- Quantitative image analysis using ImageJ, Matlab, Python…
- in vitro reconstitution of minimal, biomimetic systems: supported lipid membranes (SLBs), giant unilamellar vesicles (GUV), handling of other purified proteins.
- Micro-manipulation of GUVs (e.g. micro-pipette aspiration, optical tweezers)
- Molecular Cell Biology techniques: cell culture, transfection with plasmids to express additional proteins
- Immunofluorescence (IF) to visualise protein organisation in fixed cells.
- Biochemistry/ molecular biology techniques: western blots, protein purification…
- Fluorescence spectroscopy
- Dynamic light scattering
- Circular dichroism spectroscopy
- Chemical cross-linking / SDS PAGE

Funding Notes

Studentship includes: fees, a tax-free stipend of at least £15,009 p.a (to rise in line with UKRI recommendation); a travel allowance in year 1; a travel / conference budget; a generous consumables budget and use of a MacBook Pro for the duration of the programme. In order to apply you must ensure that you are eligible.

References

Study providing evidence for nsp3, nsp4 and nsp6 driven DMV formation in cells:
Hagemeijer, M. C., Monastyrska, I., Griffith, J., van der Sluijs, P., Voortman, J., van Bergen en Henegouwen, P. M., Vonk, A. M., Rottier, P. J. M., Reggiori, F., & De Haan, C. A. M. (2014). Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4. Virology, 458–459(1), 125–135. https://doi.org/10.1016/j.virol.2014.04.027

Study of reconstituted SARS coronavirus peptides using GUVs:
Alsaadi, E. A. J., Neuman, B. W., & Jones, I. M. (2019). A Fusion Peptide in the Spike Protein of MERS Coronavirus. Viruses, 11(9), 825. https://doi.org/10.3390/v11090825

Study of membrane-curvature sensitive protein-lipid interactions:
Brooks, R.L. and Dixon, A.M. (2020) Revealing the mechanism of protein-lipid interactions for a putative membrane curvature sensor in plant endoplasmic reticulum.

Biochim Biophys Acta Biomembr. 1862(3), 183160.

https://doi.org/10.1016/j.bbamem.2019.183160

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