The Accessory Protein ORF3a Hijacks CLCC1 Chloride Channel to Cause ER Dysfunction in Beta-coronavirus Pathogenesis

The endoplasmic reticulum (ER) homeostasis is crucial for host cells and is influenced by beta-coronaviruses upon invasion. However, the mechanisms by which viral proteins interact with ER-resident host factors to modulate ER functions and morphology remain poorly understood. The accessory protein ORF3a of SARS-CoV-2 plays a pivotal role in viral pathogenesis and modulating host immune responses. The ER-localized chloride channel CLCC1 has been identified as a strong interaction partner of ORF3a, yet the consequences of this interaction are not fully elucidated. Here, we demonstrate that ORF3a interacts with CLCC1 to modulate ER ion homeostasis, including increased ER luminal [Cl?], [K+], and decreased ER [Ca2+], and to trigger unfolded protein responses. The ORF3a-CLCC1 interaction is linked to ER phagy and nucleophagy, monitored by newly developed ratiometric reporters. Mechanistically, ORF3a induces the formation of endogenous CLCC1 puncta, while overexpression of CLCC1 attenuates ORF3a-associated toxicity by sequestering ORF3a within the ER. Furthermore, the conservation of ORF3a functions across beta-coronaviruses suggests it is a potential therapeutic target and uncovers ORF3a-mediated phenotypes spatiotemporally. In addition, ORF3a expression in mouse brains causes ER stress, ER phagy, nucleophagy, and endomembrane reorganization, shedding light on the neurological manifestations and long-term effects observed in COVID-19 patients.