SARS-CoV-2 ORF7a induces cytotoxicity associated with AP-1 signaling and ferroptosis-related responses

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), whose accessory proteins have incompletely understood roles in cellular injury. Bioinformatic analysis identified putative N-terminal signal peptides in ORF7a and ORF8. We characterized their expression, subcellular localization, and cytopathic effects in vitro. EGFP-tagged ORF7a and ORF8 showed intracellular ER–Golgi-associated distributions, and untagged ORF7a showed Golgi-associated localization. ORF7a, but not ORF8, induced prominent cytotoxicity in HEK293T cells, as assessed by real-time cell analysis and Annexin V/PI staining. Transcriptomic profiling identified changes in stress-, cell-death-, and ferroptosis-related genes. Ferrostatin-1 attenuated ORF7a-associated cytotoxicity, consistent with ferroptosis-related mechanisms. Mutation of the C-terminal ER retrieval motif (ORF7a-ARA) altered subcellular distribution and attenuated cytotoxicity and cFos/cJun accumulation. The AP-1 inhibitor T-5224 also attenuated cytotoxicity. Additional experiments demonstrated ORF7a localization in A549 cells and extended selected cytotoxicity and inhibitor-response findings to HK-2 cells. In HK-2 cells, ORF7a increased cJun abundance and reduced SLC7A11 and HO-1 protein levels. Together, these findings link the ER retrieval motif to ORF7a-associated cellular injury and support AP-1 involvement and the presence of ferroptosis-related features. Clinical trial number: not applicable.