ASC Dynamics Associated with ORF3a Protein Expression in Microvascular Lung Endothelial Cells

The NLRP3 inflammasome is a key sensor of cellular stress and pathogen-associated signals in the innate immune system. Using confocal fluorescence microscopy in microvascular lung endothelial cells, we examined ASC speck formation following expression of the SARS-CoV-2 ORF3a protein. ORF3a engaged caspase-1 through dual architectural modes: alongside the canonical pyroptosome-like pathway, a novel distributed, multifocal inflammasome pathway emerged. Multiple ASC specks were observed at later time points, yet caspase-1 activation could occur in their absence, indicating that visible speck assembly is not strictly required for inflammasome signalling. These observations provide insight into the spatiotemporal dynamics of ASC during viral protein expression. The findings contribute to a better understanding of NLRP3 inflammasome regulation under conditions of viral replication.