Keywords: Vaccinia virus assembly, Cellular membrane remodelling, Broad interactome proteomics, Protein interactions, Virus-host interactions Internship Duration: 30/11/-1 - 30/11/-1
Head of the hosting team: Emmanuelle Quemin
Website: Click here
Address of the host laboratory: Institute for Integrative Biology of the Cell Team Replication and Assembly of poxviruses 1, avenue de la Terrasse 91198 GIF SUR YVETTE CEDEX France
Supervisor: Emmanuelle QueminE-mail: emmanuelle.quemin@i2bc.paris-saclay.fr Phone: +33628015652
The assembly of Vaccinia virus (VACV) takes place in the host cytoplasm and involves a unique mechanism of membrane biogenesis via the recruitment and remodelling of cellular membranes. Vesicles derived from the endoplasmic reticulum are ruptured open into membrane intermediates with stabilized open ends, which then associate into typical open VACV-crescents. Following packaging of viral core proteins and genome into the growing crescents, the immature virions are formed after closure but need to undergo further maturation steps to form infectious particles. Such a dynamic remodelling of cellular membranes is uncommon and involves not only fission and fusion but also the formation of vesicles derived from the endoplasmic reticulum and the maintenance of hydrophobic open ends in the host cytoplasm. Five viral membrane assembly proteins or VMAPs are known to be essential for the assembly of the viral membrane of VACV. In order to characterize the role of these proteins in remodelling membranes and how they interact with each other as well as with additional partners, we would like to analyze the specific interactome of these proteins together with the Proteomics-Gif facility (https://www.i2bc.paris-saclay.fr/proteomics/proteomics-gif-sicaps/). The idea is to use chemical cross-linking and mass spectrometry. Initially, experimental conditions will be optimized (e.g. cross-linking agent, timing of incubation, sample preparation) and then, validation will be based on already reported interactions. With chemical cross-linking, protein interactions in cells will be stabilized including transient or weak intermolecular protein complexes. We will thus be able to identify currently unknown viral and cellular partners during VACV assembly (>6 hpi) for all VMAPs but also during replication (2-5 hpi) in the case of the early/late L2 specifically. If the chemical cross-linking proteomic approach is not successful, we will switch to affinity or tandem affinity purification mass spectrometry taking advantage of available recombinant VACV for which VMAPs are labelled with a GFP or HA tag.
- mammalian cell culture - work with Vaccinia virus (BSL-2) - mass-spectrometry - data analysis
https://doi.org/10.1016/j.virol.2015.02.003 https://doi.org/10.1073/pnas.1716255114
