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

View ORCID ProfileAlexandria N. Miller, Patrick R. Houlihan, View ORCID ProfileElla Matamala, View ORCID ProfileDeny Cabezas-Bratesco, View ORCID ProfileGi Young Lee, View ORCID ProfileBen Cristofori-Armstrong, Tanya L. Dilan, Silvia Sanchez-Martinez, View ORCID ProfileDoreen Matthies, Rui Yan, Zhiheng Yu, View ORCID ProfileDejian Ren, View ORCID ProfileSebastian E. Brauchi, View ORCID ProfileDavid E. Clapham
doi: https://doi.org/10.1101/2022.09.02.506428
Alexandria N. Miller
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
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  • For correspondence: millera@janelia.hhmi.org sbrauchi@uach.cl claphamd@janelia.hhmi.org
Patrick R. Houlihan
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
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Ella Matamala
2Physiology Institute, Universidad Austral de Chile, and Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD), Valdivia, 511-0566, Chile
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  • ORCID record for Ella Matamala
Deny Cabezas-Bratesco
2Physiology Institute, Universidad Austral de Chile, and Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD), Valdivia, 511-0566, Chile
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Gi Young Lee
3Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
4Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853, USA
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Ben Cristofori-Armstrong
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
5Center for Advanced Imaging, University of Queensland, St. Lucia, QLD 4072, Australia
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Tanya L. Dilan
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
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Silvia Sanchez-Martinez
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
6Molecular Biology Department, University of Wyoming, Laramie, WY, 82071, USA
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Doreen Matthies
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
7Unit on Structural Biology, Division of Basic and Translational Biophysics, Eunice Kennedy Shriver, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA
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Rui Yan
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
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Zhiheng Yu
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
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Dejian Ren
3Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
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Sebastian E. Brauchi
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
2Physiology Institute, Universidad Austral de Chile, and Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD), Valdivia, 511-0566, Chile
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  • For correspondence: millera@janelia.hhmi.org sbrauchi@uach.cl claphamd@janelia.hhmi.org
David E. Clapham
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA
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  • For correspondence: millera@janelia.hhmi.org sbrauchi@uach.cl claphamd@janelia.hhmi.org
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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 a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 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 basic 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 ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted September 03, 2022.
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The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
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
bioRxiv 2022.09.02.506428; doi: https://doi.org/10.1101/2022.09.02.506428
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The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
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
bioRxiv 2022.09.02.506428; doi: https://doi.org/10.1101/2022.09.02.506428

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