“Scientific Basis and Translational Potential:
Innate immune restriction factors such as IFITM1-3 and SERINC3/5 play crucial roles in blocking viral entry and replication, yet their precise nanoscale organization and mechanisms of action at the single-virus or single-molecule level remain poorly understood. This project leverages cutting-edge single molecule microscopy, particularly DNA-PAINT super-resolution imaging, to map the spatial distribution, stoichiometry, and dynamics of these factors in the cellular membrane and in viral particles. By achieving nanometre-scale resolution, the project will provide mechanistic insight into how these proteins are organized in host cells to inhibit viral fusion.
Techniques and Skills Development:
The student will develop expertise in super-resolution microscopy (DNA-PAINT), single-molecule localization analysis, advanced quantitative image processing, and correlative fluorescence imaging approaches. In parallel, they will gain hands-on experience in molecular cloning, cell line generation, virology techniques (e.g. pseudovirus production and infection assays), and computational data analysis using Python or MATLAB.
Overarching Aim:
To elucidate the molecular mechanisms by which IFITM1-3 and SERINC3/5 inhibit viral infection by defining their spatial organization, stoichiometry, and interactions at the nanoscale using single-molecule imaging techniques.
PhD Objectives (Years 1–4):
Year 1: Establishment of stable cell lines expressing tagged versions of IFITM1-3 and/or SERINC3/5; optimisation of DNA-PAINT labelling protocols; training in image acquisition and analysis. Objective: Validate DNA-PAINT imaging of at least one IFITM protein and quantify its distribution at the plasma membrane of host cells.
Year 2: Extend DNA-PAINT to study multiple factors simultaneously; image innate restriction factors during viral challenge; perform correlative studies with viral fusion markers. Objective: Quantify changes in protein clustering and distribution in response to viral entry.
Year 3: Determine molecular interactions and spatial coordination between IFITM during viral entry; assess effects of viral mutations or inhibitors on protein organisation. Objective: Map protein–protein proximities and infer mechanisms of inhibition during virus entry.
Year 4: Integrate findings into mechanistic models; prepare publications and thesis. Objective: Submit at least one first-author manuscript based on super-resolution datasets and prepare PhD thesis.
MRes Rotation Project (3-month option):
The rotation project will focus on establishing DNA-PAINT imaging of a single innate immune factor (e.g., IFITM3) in cell line expressing a DNA-PAINT tag. The student will learn the fundamentals of super-resolution microscopy, perform imaging and basic image analysis, and quantify protein distribution under basal and virus-challenged conditions. The outcome will be a proof-of-principle dataset demonstrating the feasibility of nanoscale imaging of innate immune effectors.”