SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling
Abstract 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 cation-selecti...
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Nature Portfolio
2025-02-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-024-07442-5 |
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| author | Antonio Michelucci Luigi Sforna Riccardo Focaia Maria Vittoria Leonardi Angela Di Battista Giorgia Rastelli Simone Vespa Simona Boncompagni Manlio Di Cristina Luigi Catacuzzeno |
| author_facet | Antonio Michelucci Luigi Sforna Riccardo Focaia Maria Vittoria Leonardi Angela Di Battista Giorgia Rastelli Simone Vespa Simona Boncompagni Manlio Di Cristina Luigi Catacuzzeno |
| author_sort | Antonio Michelucci |
| collection | DOAJ |
| description | Abstract 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 cation-selective 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. |
| format | Article |
| id | doaj-art-770c1f7bc2f344409e78e2a9dcf96795 |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-770c1f7bc2f344409e78e2a9dcf967952025-02-09T12:50:31ZengNature PortfolioCommunications Biology2399-36422025-02-018111310.1038/s42003-024-07442-5SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swellingAntonio Michelucci0Luigi Sforna1Riccardo Focaia2Maria Vittoria Leonardi3Angela Di Battista4Giorgia Rastelli5Simone Vespa6Simona Boncompagni7Manlio Di Cristina8Luigi Catacuzzeno9Department of Chemistry, Biology and Biotechnology, University of PerugiaDepartment of Chemistry, Biology and Biotechnology, University of PerugiaDepartment of Chemistry, Biology and Biotechnology, University of PerugiaDepartment of Chemistry, Biology and Biotechnology, University of PerugiaDepartment of Chemistry, Biology and Biotechnology, University of PerugiaDepartment of Neuroscience, Imaging and Clinical Sciences, University “G. d’Annunzio” of Chieti-PescaraDepartment of Medicine and Aging Sciences, University “G. d’Annunzio” of Chieti-PescaraDepartment of Neuroscience, Imaging and Clinical Sciences, University “G. d’Annunzio” of Chieti-PescaraDepartment of Chemistry, Biology and Biotechnology, University of PerugiaDepartment of Chemistry, Biology and Biotechnology, University of PerugiaAbstract 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 cation-selective 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.https://doi.org/10.1038/s42003-024-07442-5 |
| spellingShingle | Antonio Michelucci Luigi Sforna Riccardo Focaia Maria Vittoria Leonardi Angela Di Battista Giorgia Rastelli Simone Vespa Simona Boncompagni Manlio Di Cristina Luigi Catacuzzeno SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling Communications Biology |
| title | SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling |
| title_full | SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling |
| title_fullStr | SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling |
| title_full_unstemmed | SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling |
| title_short | SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling |
| title_sort | sars cov 2 orf3a accessory protein is a water permeable channel that induces lysosome swelling |
| url | https://doi.org/10.1038/s42003-024-07442-5 |
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