The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
- Janelia Research Campus, United States
- Universidad Austral de Chile, Chile
- University of Pennsylvania, United States
- Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
Abstract
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.
Data availability
All constructs and stable cell lines generated are available upon request. Atomic coordinates and cryo-EM density maps of have been deposited with the Protein Data Bank and Electron Microscopy Data Bank with the accession numbers: 8EQJ (SARS-CoV-2 Orf3a, LE/Lyso, MSP1D1 nanodisc; EMD-28538), 8EQT (SARS-CoV-2 Orf3a, PM, MSP1D1 nanodisc; EMD-28545), 8EQU (SARS-CoV-2 Orf3a, LE/Lyso, Saposin A nanodisc; EMD-28546) and 8EQS (SARS-CoV-1 Orf3a, LE/Lyso, MSP1D1 nanodisc; EMD-28544).
Article and author information
Author details
Patrick R Houlihan
Sebastian E Brauchi
Funding
National Institute of Health (GM133172,HL147379)
- Dejian Ren
Australian National Health and Medical Research Council (APP1162427)
- Ben Cristofori-Armstrong
Comisión National de Investigación de Cientifica y Tecnologíca (2117080)
- Deny Cabezas-Bratesco
Millennium Nucleus of Ion Channels -- Associated Diseases (NCN19_168)
- Sebastian E Brauchi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Publication history
- Received: October 26, 2022
- Accepted: January 25, 2023
- Accepted Manuscript published: January 25, 2023 (version 1)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
eLife 12:e84477.
https://doi.org/10.7554/eLife.84477
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