Presentation description

Previously, we showed that expression of SARS-CoV-2 Spike protein in HEK293T cells induces endoplasmic reticulum (ER) stress, activates the unfolded protein response via the PERK pathway, and triggers ER stress-induced autophagy. Given the known interplay between ER stress, autophagy, and inflammation, we investigated whether cytoplasmic spike protein expression also affects the NF-κB signaling pathway, a key regulator of inflammation typically activated through the canonical IKKα/β kinase complex.

HEK293T cells transfected with a spike protein-expressing pcDNA construct exhibited significant NF-κB activation at 48 hours post-transfection, as evidenced by IκBα degradation by western blot analysis. Unexpectedly, this activation occurred alongside marked downregulation of IKKα and IKKβ. To explore the role of autophagy, we treated HEK293T cells with Rapamycin, an autophagy inducer, which led to a reduction in IKKα but not IKKβ levels. In contrast, treatment of spike-transfected cells with low concentrations of Bafilomycin, an autophagy inhibitor, resulted in further reductions in both IKKα and IKKβ, enhanced IκBα degradation, and elevated expression of ER stress markers compared to untreated spike-transfected cells.

These findings suggest that initial autophagy inhibition by Bafilomycin exacerbates ER stress, which in turn promotes compensatory autophagy. This enhanced autophagy possibly contributes to IKKα degradation, while IKKβ downregulation appears to happen through a distinct ER stress related mechanism. Despite the downregulation of canonical NF-κB signaling components, NF-κB remained significantly activated in spike expressing cells, suggesting activation may be sustained by residual IKKα/β activity or through alternative, non-canonical pathways. Our findings indicate that spike protein-induced ER stress and autophagy modulate IKKα/β stability through mechanisms that warrant further investigation. Despite this, NF-κB activation persists, which may contribute to prolonged inflammation and provides some insight into SARS-CoV-2 associated acute kidney injury.