Skip to main content
. 2023 Jan 25;12:e84477. doi: 10.7554/eLife.84477

Figure 2. 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 CoV-2 Orf3aSNAP by doxycycline induction in various external cationic solutions (Na+, n=26; K+, n=5; Cs+, n=8; NMDG+, n=8; Ca2+, n=5). Mean traces are colored based on Figure 2A. (C) Average current density for untransfected HEK293 cells (gray bars) and cells transfected with CoV-2 Orf3aSNAP (red bars) at –80 and +80 mV recorded in Na+ (n=11), K+ (n=8), and Cs+ (n=8) solutions. (D–I) CoV-2 Orf3a does not elicit a Na+, K+, or Ca2+-selective current in endolysosomes. (D, G) Solutions used in the endolysosomal patch-clamp experiments. All the bath solutions contained 150 mM Cl- and pipette solutions contained 5 mM Cl- (E, H) I-V relationship for endolysosomes from HEK293 cells expressing GFP (control, black) or CoV-2 Orf3aHALO (green). (F, I) Average current density for control and CoV-2 Orf3aHALO expressing HEK293 cells at –120 mV and +120 mV from (D, G). (J–L) CoV-2 Orf3a does not elicit a current in Xenopus oocytes when recorded in high K+ external solution. (J–K) Representative current traces from Xenopus oocytes injected with (J) water or (K) CoV-2 Orf3a2x-STREP cRNA (20 ng). Recordings are done in high external K+ (96 mM KCl) that reproduces published methods. (L) I-V relationship for water-injected (black, n=7) or CoV-2 Orf3a (green, n=7) following protocol described in (J–K).

Figure 2.

Figure 2—figure supplement 1. SARS-CoV-2 Orf3a does not elicit a H+-selective current in endolysosomes.

Figure 2—figure supplement 1.

(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 expressing SARS-CoV-2 Orf3aHALO (CoV-2 Orf3a, blue and red traces). (C) Average current density for untransfected HEK293 cells (control) and SARS-CoV-2 Orf3aHALO (CoV-2 Orf3a) at –100 mV and +100 mV.
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.

Figure 2—figure supplement 2.

(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. White arrows indicate plasma membrane localization. (B–C) CoV-2 Orf3a does not elicit a current at the surface of A549 cells. (B) Solutions and voltage ramp protocol for A549 whole-cell patch-clamp experiments. (C) I-V relationship for A549 parental cells (yellow, n=10) and doxycycline-inducible CoV-2 Orf3aGFP stable cell line (green, n=11), both treated with doxycycline, from three independent experiments. A minimal intrinsic outward current observed in both populations is likely due to background volume regulated anion current (VRAC) conductance elicited during whole-cell access.
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.

Figure 2—figure supplement 3.

(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 constructs used for two-electrode voltage clamp studies. Western blot detecting Orf3a2x-STREP, which migrates at ~35 kDa. Total cell lysate was used as an input (filled circles). CoV-1 and CoV-2 Orf3a Xenopus oocytes surface biotinylation was detected when exposed to the biotin probe (open circles, surface biotinylation versus no biotinylation). (B) I-V relationship for water injected (black, n=7) or CoV-1 Orf3a2x-STREP mRNA injected (purple, n=7) Xenopus oocytes following protocol described in (Figure 2J–K). Water injected I-V trace is the same trace as in Figure 2L. (C–F) CoV-2 Orf3a does not elicit a current in Xenopus oocytes in ND-96 external solution (Methods). Representative current traces from Xenopus oocytes injected with (C) water, (D) CoV-1 Orf3a2x-STREP or (E) CoV-2 Orf3a2x-STREP mRNA (20 ng). Recordings are done in ND-96 solution (96 mM NaCl) following a voltage protocol that recapitulates published methods. (F) I-V relationship for water injected (black, n=7), CoV-1 Orf3a2x-STREP (purple, n=7), or CoV-2 Orf3a2x-STREP (green, n=7) following protocol described in (C–E). (G–I) Neither CoV-1 nor CoV-2 Orf3a elicits a current at the surface of HEK293 cells. (G) Solutions and voltage step protocol used for whole-cell patch-clamp experiments. (H) Representative current traces of HEK293 cells untransfected (black), or doxycycline-induced CoV-2 Orf3aSNAP (red) or CoV-1 Orf3aSNAP (blue) recorded using the voltage step protocol and solutions in (G). (I) I-V relationship for untransfected HEK293 cells (black, n=9), and cells doxycycline-induced to express CoV-2 Orf3aSNAP (red, n=11) and CoV-1 Orf3aSNAP (blue, n=9).
Figure 2—figure supplement 3—source data 1. | Raw unedited western blots and figures with the uncropped blots for Figure 2—figure supplement 3A.