SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling

Open reading frame 3a (ORF3a) is among the most expressed viral accessory proteins of SARS-CoV-2, the pathogen responsible for the last pandemic. ORF3a mainly targets lysosomes of the host cell and promotes lysosomal inactivation through their deacidification, an essential step for lysosomal exocytosis and virus egress. However, the exact mechanism through which ORF3a performs this function is still unclear. While seminal studies suggested ORF3a functioning as a cation-selective viroporin, a recent work disproved this conclusion. To unravel the possible function of ORF3a, here we employed a multidisciplinary approach including molecular dynamics (MD), molecular biology, electrophysiology, and electron microscopy. Our electrophysiological results, in accordance with apreliminary MD structural analysis, ruled out that ORF3a functions as anion channel when expressed in HEK293 cells. Conversely, both MD and videoimaging experiments for the assessment of cell volume changes demonstrated that ORF3a mediates the transmembrane transport of water. Using MD, we also identified the putative selectivity filter for water permeation, and experimentally confirmed its relevance for water transport by showing that its mutation at the level of an asparagine (N82L and N82W) abolishes ORF3a-mediated water permeation. Finally, ORF3a expression in HEK293 cells determined lysosomal volume increase (swelling), mitochondrial damage, and accumulation of intracellular membranes, and these effects were reverted by the N82W mutation. Collectively, our data suggest a new function for the ORF3a protein as a lysosomal water permeable channel. The ORF3a-mediated water transport across lysosome membrane might promote lysosomal swelling and deacidification and, by consequence, inactivation, a key step to promote virus egress from the cell.