Application of X-Ray Cryo-Tomography Technique (Cryo-XT)

VV (vaccinia virus) is one of the most complex viruses, with a size of more than 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and major rearrangements of its structural components. In this study, infected cells were selected by light fluorescence microscopy and subsequently imaged by X-ray microscopy under cryogenic conditions. Tomographic tilt series of X-ray images were used to produce three-dimensional reconstructions showing different cellular organelles (nuclei, mitochondria, ER), along with two other types of viral particles related to different stages of vaccinia virus maturation (IV) immature and (MV) mature particles; witaferin assays showed actin binding, which prevents polymerization and elongation of filaments; causing mispackaged or aberrant virions, which inhibits the progression of viral infection. The findings demonstrate that X-ray cryo-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed whole cells.