The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
- Janelia Research Campus, United States
- Physiology Institute and Millennium Nucleus of Ion Channel-Associated Diseases, Universidad Austral de Chile, Chile
- Department of Biology, University of Pennsylvania, United States
Figures
Figure 1 with 2 supplements
SARS-CoV-2 Orf3a colocalizes with markers for the plasma membrane and the endocytic pathway by live-cell imaging.
(A) Summary table of SARS-CoV-2 (CoV-2) Orf3aHALO colocalization with subcellular protein markers. All markers used to identify cellular compartments are listed in the table in A and are transiently …
Figure 1—figure supplement 1
SARS-CoV-2 Orf3a colocalizes with markers of the endocytic pathway, but not with a Golgi marker, by immunostaining.
(A) Summary table of SARS-CoV-2 (CoV-2) Orf3aHALO colocalization with subcellular antibody markers. All markers used to identify cellular compartments are listed in the table in A. (B) Fixed HEK293 …
Figure 1—figure supplement 2
SARS-CoV-1 Orf3a colocalizes with markers for the plasma membrane and the endocytic pathway by live-cell imaging.
(A) Summary table of SARS-CoV-1 (CoV-1) Orf3aHALO colocalization with subcellular protein markers. All markers used to identify cellular compartments are listed in the table (A) and are transiently …
Figure 2 with 3 supplements
SARS-CoV-2 Orf3a is not a viroporin.
(A–C) SARS-CoV-2 (CoV-2) Orf3a does not elicit a cation current at the plasma membrane. (A) Solutions used for whole-cell patch-clamp experiments. (B) I-V relationship for HEK293 cells expressing …
Figure 2—figure supplement 1
SARS-CoV-2 Orf3a does not elicit a H+-selective current in endolysosomes.
(A) Internal and external recording solutions used in the endolysosomal patch-clamp experiment. (B) I-V relationship for untransfected HEK293 cells (control, black and orange traces) and transiently …
Figure 2—figure supplement 2
SARS-CoV-2 Orf3a does not elicit a cation selective current at the plasma membrane of A549 lung alveolar cells.
(A) Plasma membrane localization of SARS-CoV-2 (CoV-2) Orf3a is observed in A549 cells. Live-cell image of CoV-2 Orf3aGFP (green) using a A549 doxycycline-inducible CoV-2 Orf3aGFP stable cell line. …
Figure 2—figure supplement 3
SARS-CoV-1 Orf3a is not a cationic ion channel at the plasma membrane of HEK293 cells and Xenopus oocytes.
(A) Surface biotinylation experiments using Xenopus oocytes injected with SARS-CoV-1 (CoV-1) Orf3a2x-STREP, SARS-CoV-2 (CoV-2) Orf3a2x-STREP, or water demonstrates PM localization of Orf3a …
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Figure 2—figure supplement 3—source data 1
| Raw unedited western blots and figures with the uncropped blots for Figure 2—figure supplement 3A.
- https://cdn.elifesciences.org/articles/84477/elife-84477-fig2-figsupp3-data1-v2.zip
Figure 3 with 5 supplements
A narrow cavity detected in the SARS-CoV-2 Orf3a TM region is unlikely to conduct cations.
(A–C) Overall architecture of SARS-CoV-2 (CoV-2) Orf3a. (A) Cryo-EM map of dimeric CoV-2 Orf3a (dark and light pink), with density for lipids colored (orange, purple). (B) Three side views of CoV-2 …
Figure 3—figure supplement 1
Cryo-EM data processing workflow for SARS-CoV-2 Orf3a reconstituted in LE/Lysosomal MSP1D1-containing nanodiscs.
Text color denotes that the program Relion 3.0 (green) or cryoSPARC v3.0 (dark blue) was used for the step of the workflow (Zivanov et al., 2018; Punjani et al., 2017; Punjani et al., 2020). Details …
Figure 3—figure supplement 2
Cryo-EM data processing workflow for SARS-CoV-2 Orf3a reconstituted in LE/Lysosomal MSP1D1-containing nanodiscs, continued from Figure 3—figure supplement 1.
Figure 3—figure supplement 3
Structural determination of SARS-CoV-2 Orf3a LE/Lyso (A–D) or PM (E–H) MSP1D1 nanodiscs.
(A, E) Angular orientation distributions of particles used in the final reconstructions. The particle distributions are indicated by color shading, with blue to red representing low and high numbers …
Figure 3—figure supplement 4
Cryo-EM data processing workflow for SARS-CoV-2 Orf3a reconstituted in PM MSP1D1-containing nanodiscs.
Text color denotes the program Relion 3.1 (green) or cryoSPARC v3.0 (dark blue) (Zivanov et al., 2018; Punjani et al., 2017; Punjani et al., 2020). Details are described in the Methods. …
Figure 3—figure supplement 5
Representative cryo-EM density for SARS-CoV-2 Orf3a and SARS-CoV-1 Orf3a structures.
(A–D) Four representative areas of cryo-EM density (blue mesh) from the four Orf3a datasets with structures represented as sticks and colored as follows: SARS-CoV-2 Orf3a LE/Lyso MSP1D1-containing …
Figure 4 with 5 supplements
Two SARS-CoV-2 Orf3a lateral openings within the TM region are filled with density likely representing lipid sites.
(A) Two side views of SARS-CoV-2 (CoV-2) Orf3a in LE/Lyso MSP1D1 nanodiscs highlighting two subunits (dark and light pink). Lipid densities (blue mesh, contoured at 7σ) are identified in …
Figure 4—figure supplement 1
Comparison of lipid densities between (A, D) SARS-CoV2 Orf3a LE/Lyso MSP1D1-containing nanodiscs, (B, E) SARS-CoV2 Orf3a LE/Lyso Saposin A-containing nanodiscs, and (C, F) SARS-CoV-1 Orf3a LE/Lyso MSP1D1-containing nanodiscs.
Side views (A–C) and cutaway views from the extracellular/luminal side of the membrane (D–F). Density for Lipid Site 1 (orange) and Lipid Site 2 (purple) highlights distinct binding between …
Figure 4—figure supplement 2
Cryo-EM data processing workflow for SARS-CoV-2 Orf3a reconstituted in LE/Lyso Saposin A-containing nanodiscs.
Text color denotes that the program Relion 3.1 (green) or cryoSPARC v3.0 (dark blue) (Zivanov et al., 2018; Punjani et al., 2017; Punjani et al., 2020). Details are described in the Methods. The …
Figure 4—figure supplement 3
Structural determination of SARS-CoV-2 Orf3a LE/Lyso Saposin A nanodisc (A–D) or SARS-CoV-1 LE/Lyso MSP1D1 nanodisc (E–H).
(A, E) Angular orientation distributions of particles used in the final reconstructions. The particle distributions are indicated by color shading, with blue to red representing low and high numbers …
Figure 4—figure supplement 4
Cryo-EM data processing workflow for SARS-CoV-1 Orf3a reconstituted in LE/Lysosomal MSP1D1-containing nanodiscs.
Text color denotes the program Relion 3.1 (green) or cryoSPARC v3.0 (dark blue) (Zivanov et al., 2018; Punjani et al., 2017; Punjani et al., 2020) Details are described in the Methods.
Figure 4—figure supplement 5
A similar narrow cavity is detected in the TM region of SARS-CoV-1 Orf3a.
(A) Two representative side views of SARS-CoV-1 (CoV-1) Orf3a in LE/Lyso MSP1D1-containing nanodiscs highlighting two subunits (dark and light green). Inspection of the TM region for a pore, …
Figure 5 with 2 supplements
SARS-CoV-2 Orf3a does not elicit ion flux or conductances in a vesicle-reconstituted system.
(A) Schematic of the ACMA-based fluorescence flux assay (Zhang et al., 1994; Heginbotham et al., 1998; Miller and Long, 2012; Kane Dickson et al., 2014). A K+ (pink) or Cl- (blue) gradient is …
Figure 5—figure supplement 1
Characterization of vesicle-reconstituted SARS-CoV-1 and SARS-CoV-2 Orf3a at low and high protein ratios.
(A–B) K+ (A) or Cl- (B) flux is not observed in SARS-CoV-2 (CoV-2) Orf3a2x-STREP-reconstituted vesicles (blue) as compared with the empty vesicle control (black, n=3) using 1:10 (n=3) (A) or 1:25 …
Figure 5—figure supplement 2
Multiple conductance species are observed from SARS-CoV-2 Orf3a containing-vesicles reconstituted at a high protein to lipid ratio and likely result from transient membrane leakiness and/or contamination by bona fide ion channels.
(A) Symmetrical recording solutions with CaCl2 (green), KCl (blue) or NaCl (orange) used for proteoliposome patch-clamp experiments. (B) Multiple K+, Na+ and Ca2+ conductance species are observed by …
Figure 6 with 1 supplement
SARS-CoV-2 Orf3a, but not SARS-CoV-1 Orf3a, interacts with HOPS protein, VPS39.
(A–B) Rab7 puncta (green) are abundant in HEK293 cells expressing (A) SARS-CoV-2 (CoV-2) Orf3aHALO (magenta), but not (B) SARS-CoV-1 (CoV-1) Orf3aHALO (magenta; Hoechst 33342, blue). (C) …
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Figure 6—source data 1
Raw unedited western blots and figures with the uncropped blots for Figure 6C.
- https://cdn.elifesciences.org/articles/84477/elife-84477-fig6-data1-v2.zip
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Figure 6—source data 2
Raw unedited western blots and figures with the uncropped blots for Figure 6F.
- https://cdn.elifesciences.org/articles/84477/elife-84477-fig6-data2-v2.zip
Figure 6—figure supplement 1
Purification of SARS-CoV-1 and SARS CoV-2 Orf3a loop chimeras.
(A, C) Gel filtration traces from (A) SARS-CoV-2 Orf3a loop chimera (CoV-2 Orf3a LC) and (C) SARS-CoV-1 Orf3a loop chimera (CoV-1 Orf3a LC) after elution from Strep-Tactin XT column. Collected peak …
Figure 7
A region of VPS39 and SARS-CoV-2 Orf3a interaction.
(A) Cytosolic view of SARS-CoV-2 Orf3a structure (dark and light pink) with the unstructured loop highlighted in yellow. W193 (purple, sticks) has also been described to mediate an interaction …
Author response image 1
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/84477/elife-84477-mdarchecklist1-v2.docx
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Supplementary file 1
Cryo-EM data collection, refinement, and validation statistics.
- https://cdn.elifesciences.org/articles/84477/elife-84477-supp1-v2.docx
