Viral Assembly and Membrane Domain Dynamics – FLUOBUDS

Retroviruses are enveloped RNA viruses responsible for many human diseases. Assembly of retroviruses mainly occurs at the cell surface upon the oligomerization of a structural viral protein, namely Gag. Gag anchors the cellular membrane via its N-terminus, namely Matrix (MA), which is myristoylated and highly enriched in basic residues. MA interacts with the plasma membrane thanks to specific interaction with the phosphoinositide PI(4,5)P2 (or PIP2) localized at the inner leaflet of this membrane. This project aims at characterizing the role of pre-existing or generated lipid nanodomains at the host cell plasma membrane during viral assembly, and defining lipid-viral protein interaction for new antiviral strategy. For correctly understanding viral assembly at the level of viral protein-lipid interaction, the project requires the production and purification of recombinant MA and Gag proteins and the help of new fluorescent lipidic probes. These new probes will be synthesized by the chemists of ENS Lyon with specifically designed chromophores. To date, commercial fluorescent PIP2 available are labelled on the acyl chain or the polar head of the lipid.This is a limit of these dyes in term of correct partitioning into the membrane nanodomains and or interacting with the proteins. This clearly justify the development of new fluorescent PIP2 bearing chromophores directly on the 3’ terminus of the glycerol part, therefore leaving free the two fatty acid chains and the polar head of the phospholipid which participate in Gag-PIP2 membrane interactions. These new fluorescent probes will be tested on biomimetic membranes and on eukaryotic cells for their toxicity, fluorescence efficiency and their capacity to integrate membranes. We will characterize Gag (or MA)/PIP2 interactions on biomimetic membranes and in cellulo, by estimating the capacity of Gag proteins (wild-type or mutants) to assemble on cellular lipid nanodomains fluorescently labelled or to generate PIP2 nanodomains in living cells using biophotonic approaches (svFCS, xICS sptPALM, FLIM). One objective of this project is to synthesize new fluorescent PIP2 probes in order to achieve lipid dynamic live cell imaging studies, at the nanoscale level, in the context of virus assembly. The second objective is to characterize proteic and lipidic determinants involved in retrovirus assembly for new antiviral approaches. This project at the interface of chemistry, biophysics, virology and cellular biology will bring new insights on macromolecular fluorescent PIP2 probes, PIP2 membrane dynamics, protein-PIP2 interaction on viral assembly mechanism of enveloped RNA viruses that bud from the plasma membrane, and ultimately, will lead to the design of new antiviral drug that will target virus-lipid interactions.