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. 2023 Jan 25;12:e84477. doi: 10.7554/eLife.84477

Figure 5. 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 generated by reconstitution and dilution into an appropriate external salt solution (K+ efflux: 150 KCl in, 150 NMDG-Cl out; Cl- flux: 110 Na2SO4 in, 125 NaCl out; in mM). If CoV-2 Orf3a conducts K+ or Cl- ions, then the addition of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) will drive H+ (green) influx. ACMA is quenched and sequestered in vesicles at low pH, resulting in loss of ACMA fluorescence. Valinomycin (Val), a K+ permeable ionophore, is added to the end of the K+ flux assay to empty all vesicles. Created with Biorender.com. (B–C) K+ (n=4) (B) or Cl- (n=4) (C) flux is not observed in SARS-CoV-2 (CoV-2) Orf3a2x-STREP-reconstituted vesicles (blue) as compared with the empty vesicle control (black, n=4) using vesicles reconstituted at a 1:100 (wt:wt) protein to lipid ratio. CCCP and Val are added as indicated (arrows). Error is represented as SEM. (D) Probability of observing an open event in a CoV-2 Orf3a2x-STREP-reconstituted proteoliposome patch with vesicle reconstituted at a 1:100 protein to lipid ratio. NaCl, n=27; KCl, n=32, and CaCl2, n=105. Error is represented as SEM.

Figure 5.

Figure 5—figure supplement 1. Characterization of vesicle-reconstituted SARS-CoV-1 and SARS-CoV-2 Orf3a at low and high protein ratios.

Figure 5—figure supplement 1.

(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 (n=3) (B) ratios of protein to lipids. The protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and K+-permeable ionophore valinomycin (Val) are added as indicated (arrows). Error is represented as SEM. (C) K+ flux is not observed in SARS-CoV-1 Orf3a2x-STREP-reconstituted vesicles (blue) as compared with the empty vesicle control (black, n=3) using a 1:25 (purple, n=3) or 1:100 (green, n=3) ratio of protein to lipids. Error is represented as SEM. (D) Schematic of 90° light-scattering K+ flux assay (Brammer et al., 2014; Stockbridge et al., 2012). CoV-2 Orf3a vesicles reconstituted in 200 mM K-glutamate are diluted into a hypertonic buffer containing 260 mM K-thiocyanate, resulting in vesicle shrinkage. If CoV-2 Orf3a is a K+-selective viroporin, then the asymmetrical K+ concentration should drive K+ influx, leading to water absorption, vesicle swelling (green arrow) and a reduction of 90° light-scattering (green line, inset). If CoV-2 Orf3a is not a K+-selective viroporin, then vesicles will not swell (gray arrow) and no change in 90° light-scattering should be observed (gray line, inset). The addition of Val (dotted line) leads to vesicle swelling and reduction of 90° light-scattering should be observed for all vesicles in the sample. Created with Biorender.com. (E–G) No difference in normalized K+ influx is observed among SARS-CoV-2 Orf3a2x-STREP reconstituted using 1:10 (E, black) or 1:100 (F, red) ratio of protein to lipids, and control vesicles (G, blue).
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.

Figure 5—figure supplement 2.

(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 proteoliposome patch-clamp with SARS-CoV-2 (CoV-2) Orf3a2x-STREP-containing vesicles reconstituted at a 1:10, but not 1:100 (Figure 5E), ratio of protein to lipid. (C) Probability of observing an open event in a CoV-2 Orf3a2x-STREP reconstituted proteoliposome patch with vesicles reconstituted at a 1:10 ratio of protein to lipid. NaCl, n=18; KCl, n=114; and CaCl2, n=35. Error is presented as SEM. (D) Table of the top 5 proteins with 2 TM helices or greater from mass spectrometry analysis of CoV-2 Orf3a2x-STREP containing vesicles reconstituted at a 1:10 protein to lipid ratio. (E) Addition of 100 μM 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic Acid (DIDS; orange trace), a blocker of VDAC channels, eliminates the Ca2+ currents observed with proteoliposome patches-reconstituted CoV-2 Orf3a2x-STREP, whereas DMSO has no effect (blue). (F) Open probability of proteoliposome patches-reconstituted CoV-2 Orf3a2x-STREP sample following protocol from (E).