A single Omicron mutation reshapes ORF3a-driven host-cell remodelling

SARS-CoV-2 ORF3a remodels host membranes, but the structural basis and metabolic consequences of this process remain unclear. Here, we combine complementary imaging approaches to define ORF3a function at nanometric scale, identifying underlying mechanisms, and determining how Omicron variant rewire this activity. ORF3a from the ancestral Wuhan strain disrupts Golgi cisternae, drives the formation of ORF3a dense vesicles, remodels mitochondrial architecture, and promotes lipid droplet expansion. Multi-omics analyses further reveal selective triacylglycerol accumulation linked to DGAT1 upregulation, which we validate pharmacologically through DGAT1 inhibition. In contrast, Omicron ORF3a variant, despite carrying only the Thr223Ile substitution within the {beta}7-{beta}8 loop at the bottom of the cytosolic domain, induced a dramatic phenotypic shift: ORF3a localizes to multivesicular bodies, preserves Golgi architecture, and fails to induce lipid accumulation. All together, these results identify ORF3a as a regulator of membrane organization and lipid homeostasis, showing how minimal sequence variation rewires host-cell remodelling. Graphical TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/742305v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@135032borg.highwire.dtl.DTLVardef@1633d4borg.highwire.dtl.DTLVardef@4d4115org.highwire.dtl.DTLVardef@1ebdf65_HPS_FORMAT_FIGEXP M_FIG C_FIG