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

Michelucci et al., Communications Biology, doi:10.1038/s42003-024-07442-5
Feb 2025  
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In Vitro and In Silico study showing that SARS-CoV-2 ORF3a accessory protein functions as a water-permeable channel that induces lysosome swelling, rather than a cation-selective viroporin as previously thought. Authors demonstrate through patch-clamp experiments that ORF3a does not mediate ion or proton currents. Instead, videoimaging experiments show ORF3a expression increases the rate of cell swelling under hypotonic conditions, indicating enhanced water permeability. Molecular dynamics simulations confirm that the tetrameric ORF3a structure allows water passage, with a selectivity filter formed by asparagine residues N82 and N119. Mutation of N82 abolishes ORF3a-mediated water transport. Electron microscopy reveals that ORF3a expression leads to enlarged lysosomes, an effect reversed by the N82W mutation. The authors propose that ORF3a-mediated water transport into lysosomes causes their swelling and deacidification, key steps in promoting SARS-CoV-2 egress through the lysosomal exocytotic pathway.
Michelucci et al., 4 Feb 2025, peer-reviewed, 10 authors. Contact: antonio.michelucci@unipg.it, luigi.catacuzzeno@unipg.it.
In Vitro studies are an important part of preclinical research, however results may be very different in vivo.
This PaperMiscellaneousAll
SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling
Antonio Michelucci, Luigi Sforna, Riccardo Focaia, Maria Vittoria Leonardi, Angela Di Battista, Giorgia Rastelli, Simone Vespa, Simona Boncompagni, Manlio Di Cristina, Luigi Catacuzzeno
Communications Biology, doi:10.1038/s42003-024-07442-5
ORF3a, the most abundantly expressed accessory protein of SARS-CoV-2, plays an essential role in virus egress by inactivating lysosomes through their deacidification. However, the mechanism underlying this process remains unclear. While seminal studies suggested ORF3a being a cationselective channel (i.e., viroporin), recent works disproved this conclusion. To unravel the potential function of ORF3a, here we employed a multidisciplinary approach including patch-clamp electrophysiology, videoimaging, molecular dynamics (MD) simulations, and electron microscopy. Preliminary structural analyses and patch-clamp recordings in HEK293 cells rule out ORF3a functioning as either viroporin or proton (H + ) channel. Conversely, videoimaging experiments demonstrate that ORF3a mediates the transmembrane transport of water. MD simulations identify the tetrameric assembly of ORF3a as the functional water transporter, with a putative selectivity filter for water permeation that includes two essential asparagines, N82 and N119. Consistent with this, N82L and N82W mutations abolish ORF3a-mediated water permeation. Finally, ORF3a expression in HEK293 cells leads to lysosomal volume increase, mitochondrial damage, and accumulation of intracellular membranes, all alterations reverted by the N82W mutation. We propose a novel function for ORF3a as a lysosomal water-permeable channel, essential for lysosome deacidification and inactivation, key steps to promote virus egress.
All relevant information supporting the findings of this study are presented in the manuscript and in Supplementary Information. Source data underlying graphs and structure and topology files resulting from MD simulations can be found in Supplementary Data. Additional supporting data is available from the corresponding authors upon request. Author contributions A.M. and L.C. conceived and designed the study and wrote the manuscript. A.M. and L.S. performed all the electrophysiological and videoimaging experiments, analyzed the results, and prepared the figures. R.F., M.V.L., and A.DiB. performed cell culture preparations and transfections and obtained immunofluorescence images; L.C. performed MD simulations and analysis; G.R. prepared samples for EM analysis; S.V. sorted EGFP positive cells used for EM preparations and edited the manuscript; S.B. performed EM experiments, analyzed results, prepared figures, and edited the manuscript; M.DiC. designed all the plasmids used in the study, supervised all molecular biology preparations, and edited the manuscript. Competing interests The authors declare no competing interests.
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