The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that nei...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2023-01-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/84477 |
| _version_ | 1811165052814753792 |
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| author | Alexandria N Miller Patrick R Houlihan Ella Matamala Deny Cabezas-Bratesco Gi Young Lee Ben Cristofori-Armstrong Tanya L Dilan Silvia Sanchez-Martinez Doreen Matthies Rui Yan Zhiheng Yu Dejian Ren Sebastian E Brauchi David E Clapham |
| author_facet | Alexandria N Miller Patrick R Houlihan Ella Matamala Deny Cabezas-Bratesco Gi Young Lee Ben Cristofori-Armstrong Tanya L Dilan Silvia Sanchez-Martinez Doreen Matthies Rui Yan Zhiheng Yu Dejian Ren Sebastian E Brauchi David E Clapham |
| author_sort | Alexandria N Miller |
| collection | DOAJ |
| description | The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a’s ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion. |
| first_indexed | 2024-04-10T15:31:20Z |
| format | Article |
| id | doaj.art-7aea50eabcf9434b9dd126c619c9efbc |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-10T15:31:20Z |
| publishDate | 2023-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-7aea50eabcf9434b9dd126c619c9efbc2023-02-13T17:05:18ZengeLife Sciences Publications LtdeLife2050-084X2023-01-011210.7554/eLife.84477The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteinsAlexandria N Miller0https://orcid.org/0000-0003-3986-7923Patrick R Houlihan1https://orcid.org/0000-0002-2505-2347Ella Matamala2Deny Cabezas-Bratesco3Gi Young Lee4Ben Cristofori-Armstrong5Tanya L Dilan6https://orcid.org/0000-0002-3944-8385Silvia Sanchez-Martinez7Doreen Matthies8https://orcid.org/0000-0001-9221-4484Rui Yan9Zhiheng Yu10Dejian Ren11Sebastian E Brauchi12https://orcid.org/0000-0002-8494-9912David E Clapham13https://orcid.org/0000-0002-4459-9428Janelia Research Campus, Ashburn, United StatesJanelia Research Campus, Ashburn, United StatesPhysiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Valdivia, ChilePhysiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Valdivia, ChileDepartment of Biology, University of Pennsylvania, Philadelphia, United StatesJanelia Research Campus, Ashburn, United StatesJanelia Research Campus, Ashburn, United StatesJanelia Research Campus, Ashburn, United StatesJanelia Research Campus, Ashburn, United StatesJanelia Research Campus, Ashburn, United StatesJanelia Research Campus, Ashburn, United StatesDepartment of Biology, University of Pennsylvania, Philadelphia, United StatesJanelia Research Campus, Ashburn, United States; Physiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Valdivia, ChileJanelia Research Campus, Ashburn, United StatesThe severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a’s ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.https://elifesciences.org/articles/84477SARS-CoVOrf3aendocytic traffickingVPS39ion channelHOPS complex |
| spellingShingle | Alexandria N Miller Patrick R Houlihan Ella Matamala Deny Cabezas-Bratesco Gi Young Lee Ben Cristofori-Armstrong Tanya L Dilan Silvia Sanchez-Martinez Doreen Matthies Rui Yan Zhiheng Yu Dejian Ren Sebastian E Brauchi David E Clapham The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins eLife SARS-CoV Orf3a endocytic trafficking VPS39 ion channel HOPS complex |
| title | The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins |
| title_full | The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins |
| title_fullStr | The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins |
| title_full_unstemmed | The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins |
| title_short | The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins |
| title_sort | sars cov 2 accessory protein orf3a is not an ion channel but does interact with trafficking proteins |
| topic | SARS-CoV Orf3a endocytic trafficking VPS39 ion channel HOPS complex |
| url | https://elifesciences.org/articles/84477 |
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