- 14 Avr 2023
- 11h00 - 0h00
Studying Vaccinia virus infection using cellular cryo electron tomography – Emmanuelle Quemin (I2BC, CNRS Gif-sur-Yvette)
Poxviruses form a diverse group of enveloped, large cytoplasmic dsDNA viruses that have epidemic potential. Since its successful use as a live vaccine to eradicate Variola virus, the etiologic agent of smallpox, Vaccinia virus (VACV) has become a model for poxvirus research. Early stages of VACV assembly rely on a unique membrane acquisition mechanism: there is no budding or envelopment at cellular compartments but instead, cellular membranes are recruited and subsequently ruptured-open to form viral membrane precursors with stabilized open ends in the host cytoplasm. These open-ended membranes are termed crescents because they associate with the scaffold protein that induces a characteristic curvature. After packaging of the viral genome into the growing crescents, spherical immature virions are formed and need to undergo further maturation to make infectious viral particles that have a brick-like morphology. Fundamental questions remain opened about how cellular membranes are recruited and ruptured-open, how the open membrane ends of crescents are stabilized in the cytoplasm, how crescents grow, and how packaging of viral DNA is regulated. While mutagenesis studies have identified key viral proteins essential for some of these processes, the underlying molecular mechanisms remain unclear. We aim to gain a better mechanistic understanding of VACV membrane assembly directly in cellula using cryo electron tomography. I will here present an optimized workflow that we have developed to target the viral assembly sites in infected cells directly using correlative methods and focused ion beam (FIB)-milling. We could so far observe viral assembly intermediates at various stages in the native cellular context with the goal of providing novel insights into the unique mechanism of poxvirus assembly.