SARS-CoV-2 Viroporins Activate The NLRP3-Inflammasome Via The Mitochondrial Permeability Transition Pore

Cytokine storm precipitated by activation of the host innate immune defenses is a major cause of COVID19 death. To elucidate how SARS-CoV-2 initiates this inflammatory process, we studied viroporin proteins E and Orf3a (2-E+2-3a). Expression of 2-E+2-3a in human 293T cells resulted in increased cytosolic Ca++ and then elevated mitochondrial Ca++, taken up through the MUCi11-sensitive mitochondrial calcium uniporter (MCU). Increased mitochondrial Ca++ resulted in stimulation of mitochondrial reactive oxygen species (mROS) production, which was blocked by mitochondrially-targeted catalase or MnTBAP. To determined how mROS activates the inflammasome, we transformed 293T cells with NLRP3, ASC, pro-caspase-1 and pro-IL-1β plus used THP1 derived macrophages to monitor the secretion of mature IL-1β. This revealed that mROS activates a factor that is released via the NIM811-sensitive mitochondrial permeability pore (mtPTP) to activate the inflammasome. Hence, interventions targeting mROS and the mtPTP may mitigate the severity of COVID19 cytokine storms. ### Competing Interest Statement DECLARATION OF INTERESTS D.C.W. serves of the advisory boards of Plano Therapeutics, Medical Excellent Capital, and has a grant from March Therapeutics.