Accurate localization of SARS-CoV-2 ORF3a in cellular sub-compartments

Viral accessory proteins are believed to play roles in the viral life cycle and may contribute to the pathogenesis and virulence. We have developed in vitro models and implemented tools that include bronchoalveolar A549 epithelial and monocyte THP1 cells individually expressing accessory proteins by lentivirus transduction. SARS-CoV-2 ORF3a is a unique membrane protein with unclear localization and still under discussion function. Our tomograms obtained at MISTRAL through previous proposals (2022025607, 2023027348) have revealed a catastrophic cellular landscape (in both, A549 and THP1 cells), with a high percentage of seriously affected mitochondria, an unusual increase of lipid drops, almost no signs of Golgi or ER and, an increased number of vesicles (some of them with tubular structures). Here we propose to use cryo correlative microscopy (cryo3DSIM combined with cryo-SXT) for the accurate determination of the topological relationship between GFP-labelled ORF3a with the distribution of organelles inside the cells compared to GFP transduced control cells