The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins

  1. Alexandria N Miller  Is a corresponding author
  2. Patrick R Houlihan
  3. Ella Matamala
  4. Deny Cabezas-Bratesco
  5. Gi Young Lee
  6. Ben Cristofori-Armstrong
  7. Tanya L Dilan
  8. Silvia Sanchez-Martinez
  9. Doreen Matthies
  10. Rui Yan
  11. Zhiheng Yu
  12. Dejian Ren
  13. Sebastian E Brauchi
  14. David E Clapham  Is a corresponding author
  1. Janelia Research Campus, United States
  2. Universidad Austral de Chile, Chile
  3. University of Pennsylvania, United States
  4. 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

  1. Alexandria N Miller

    Janelia Research Campus, Ashburn, United States
    For correspondence
    millera@janelia.hhmi.org
    Competing interests
    The authors declare that no competing interests exist.
  2. Patrick R Houlihan

    Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2505-2347
  3. Ella Matamala

    Physiology Institute, Universidad Austral de Chile, Valdivia, Chile
    Competing interests
    The authors declare that no competing interests exist.
  4. Deny Cabezas-Bratesco

    Physiology Institute, Universidad Austral de Chile, Valdivia, Chile
    Competing interests
    The authors declare that no competing interests exist.
  5. Gi Young Lee

    Department of Biology, University of Pennsylvania, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ben Cristofori-Armstrong

    Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Tanya L Dilan

    Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3944-8385
  8. Silvia Sanchez-Martinez

    Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Doreen Matthies

    Division of Basic and Translational Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9221-4484
  10. Rui Yan

    Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Zhiheng Yu

    Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Dejian Ren

    Department of Biology, University of Pennsylvania, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Sebastian E Brauchi

    Physiology Institute, Universidad Austral de Chile, Valdivia, Chile
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8494-9912
  14. David E Clapham

    Janelia Research Campus, Ashburn, United States
    For correspondence
    claphamd@hhmi.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4459-9428

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

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Publication history

  1. Received: October 26, 2022
  2. Accepted: January 25, 2023
  3. 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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  1. Alexandria N Miller
  2. Patrick R Houlihan
  3. Ella Matamala
  4. Deny Cabezas-Bratesco
  5. Gi Young Lee
  6. Ben Cristofori-Armstrong
  7. Tanya L Dilan
  8. Silvia Sanchez-Martinez
  9. Doreen Matthies
  10. Rui Yan
  11. Zhiheng Yu
  12. Dejian Ren
  13. Sebastian E Brauchi
  14. David E Clapham
(2023)
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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