Séminaire "IBS jeunes chercheurs" : Investigating poxvirus assembly using cryo electron tomography and correlative workflows

Par Dr Emmanuelle Quemin, Institut de Biologie Intégrative de la Cellule (I2BC), Gif-sur-Yvette

Poxviruses are enveloped, large cytoplasmic dsDNA viruses with epidemic potential. Vaccinia virus (VACV) has been used as a live vaccine to eradicate smallpox and is a well-established poxvirus model system. During poxvirus morphogenesis, membrane acquisition follows an unconventional mechanism without budding or wrapping. Instead, cellular membranes are being recruited to the cytoplasmic viral factory where they get ruptured and extensively remodeled to make viral membrane precursors with stabilized open ends. Subsequent binding to the viral scaffold protein induces a specific membrane curvature and the characteristic crescent shape. Membrane crescents further grow into spherical particles enclosing the viral DNA and proteins. Subsequently, the immature particles formed need to undergo further maturation steps including phosphorylation events and proteolytic cleavages to make infectious mature virions with the characteristics ovoid or brick-like shape that contain the internal core structure with a multi-layer architecture. However, the molecular mechanisms underlying these assembly steps remain poorly understood for all poxviruses. In my group, we are employing multi-modal imaging modalities including cryo electron tomography (cryoET) and correlative approaches to gain a better understanding of the stepwise VACV assembly to make large and pleomorphic infectious viral particles. For this, we are targeting viral factories by correlative imaging and focused ion beam (FIB)- milling to prepare thin lamellae from VACV-infected cells. For data collection, we combine low magnification with high-resolution tomograms in the View Tomo framework to provide key mesoscale information of the cellular context. Notably, this gives us access to : (i) the re-organization of VACV-infected cells, (ii) the viral factories within, and (iii) individual viral particles at all stages of assembly directly in their native cellular environment. Focusing on morphogenesis of the viral membrane, sub-tomogram averaging is then applied to investigate the arrangement of the viral scaffold protein on crescents and immature virions in situ with unprecedented details at the molecular and structural level.
Altogether, the imaging workflows implemented in combination with tailored processing pipelines to probe the mechanistic basis of poxvirus assembly provide significant novel insights into viral morphogenesis and are also more broadly applicable.

Hôte : Comité organisateur des séminaires " IBS Jeunes chercheurs "