SARS-CoV-2 Delta variant-specific ORF3a mutations destabilize lysosomal homeostasis to trigger lysosomal damage-mediated cell death

The variants of concern (VOCs) of SARS-CoV-2 emerged independently and became dominant globally at different times. Despite the emergence of multiple VOCs, the Delta variant of SARS-CoV-2 showed heightened pathogenicity and unprecedented mortality. However, the Delta variant specific mechanisms underlying its increased pathogenicity are unclear. Here, we show that variations in SARS-CoV-2 ORF3a correlate with the pathogenic potential of VOCs and drive Delta variant-specific lysosomal damage that activates inflammatory cell death. ORF3a from the Delta variant shows unique mutational patterns distinct from other SARS-CoV-2 VOCs. ORF3a-specific phylogenetic analysis reveals noticeable differences in the evolutionary trajectories of VOCs, likely reflective of their pathogenic relatedness. Unlike the Omicron variant and the ancestral Wuhan strain, Delta-specific ORF3a mutations promotes robust lysosomal damage, peripheral distribution, and membrane localization with no apparent effect on viral titers. Furthermore, Delta ORF3a mutations-induced lysosomal damage promotes both apoptosis and necroptosis activation in human cells. Structurally, these Delta variant-specific mutations appear to stabilize the ORF3a oligomers through helical packing and formation of a non-native disulfide bond, possibly facilitating their lysosomal association and damage. Overall, our observations indicate that ORF3a disrupts lysosomal homeostasis and triggers cell death, suggesting Delta-variant-specific regulation of cell fate and inflammation-associated pathogenesis. SARS-CoV-2 ORF3a variations from the Delta variant differentiate it from other variants of concern; these mutations promote lysosome damage and multimodal cell death activation associated with disease severity.