p2 virus  (Expression Systems Inc)

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Summary table of SARS-CoV-2 (CoV-2) Orf3a HALO colocalization with subcellular antibody markers. All markers used to identify cellular compartments are listed in the table in A. ( B ) Fixed HEK293 cells without (Dox -) and with (Dox +) doxycycline to induce expression of CoV-2 Orf3a HALO (magenta). Cell nuclei are visualized with Hoechst 33342 stain (blue). ( C–F ) Fixed HEK293 cells treated with doxycycline to induce expression of CoV-2 Orf3a HALO (magenta) stained with ( C ) EEA1, ( D ) Rab7, ( E ) LAMP1, and ( F ) TGN46 (green). Nuclei are labeled with Hoechst 33342 (blue). All images are representative of three independent experiments.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Expressing, Staining, Labeling

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Summary table of SARS-CoV-1 (CoV-1) Orf3a HALO colocalization with subcellular protein markers. All markers used to identify cellular compartments are listed in the table ( A ) and are transiently expressed (mEm, mEmerald; mNG, mNeonGreen; GFP, green fluorescent protein). ( B ) Live-cell imaging of HEK293 cells without (Dox -) and with (Dox +) doxycycline to induce expression of CoV-1 Orf3a HALO (magenta). Cell nuclei are visualized with Hoechst 33342 stain. ( C–I ) Live-cell image of transiently expressed ( C ) farnesylated-GFP, ( D ) Rab5-mEm, ( E ) Rab7-mEm, ( F ) LAMP1-mEm, ( G ) Sec61-mEm ( H ) αMannosidase-II-mEm or ( I ) Pex11-mNG (green) with CoV-1 Orf3a HALO (magenta) using the HEK293 stable cell line in ( B ). White arrows and boxes indicate regions of co-localization. All images are representative of three independent experiments.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Live Cell Imaging, Expressing, Staining, Stable Transfection

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Summary table of SARS-CoV-2 (CoV-2) Orf3a HALO colocalization with subcellular protein markers. All markers used to identify cellular compartments are listed in the table in A and are transiently expressed (mEm, mEmerald; mNG, mNeonGreen; GFP, green fluorescent protein). ( B ) Live-cell image of transiently expressed farnesylated-GFP (green) and CoV-2 Orf3a HALO (magenta) using a HEK293 doxycycline-inducible CoV-2 Orf3a HALO stable cell line. White arrows indicate co-localization. ( C ) Total Internal Reflection Fluorescence (TIRF) imaging of HEK293 cell with transient expression of CoV-2 Orf3a HALO (white). Orange box, magnification of the surface to highlight CoV-2 Orf3a HALO (black). ( D–I ) Live-cell image of transiently expressed ( D ) Rab5-mEm, ( E ) Rab7-mEm, ( F ) LAMP1-mEm, ( G ) Sec61-mEm, ( H ) αMannosidase-II-mEm, or ( I ) Pex11-mNG (green) with CoV-2 Orf3a HALO (magenta) as described in ( B ). White boxes indicate regions of co-localization. All confocal images are representative of three to six independent experiments.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Stable Transfection, Fluorescence, Imaging, Expressing

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 Orf3a SNAP by doxycycline induction in various external cationic solutions (Na + , n=26; K + , n=5; Cs + , n=8; NMDG + , n=8; Ca 2+ , n=5). Mean traces are colored based on . ( C ) Average current density for untransfected HEK293 cells (gray bars) and cells transfected with CoV-2 Orf3a SNAP (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 Ca 2+ -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 Orf3a HALO (green). ( F, I ) Average current density for control and CoV-2 Orf3a HALO 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 Orf3a 2x-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 ).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Clinical Proteomics, Membrane, Patch Clamp, Expressing, Transfection, Transferring, Control, Injection

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 Orf3a HALO (CoV-2 Orf3a, blue and red traces). ( C ) Average current density for untransfected HEK293 cells (control) and SARS-CoV-2 Orf3a HALO (CoV-2 Orf3a) at –100 mV and +100 mV.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Patch Clamp, Control, Expressing

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Plasma membrane localization of SARS-CoV-2 (CoV-2) Orf3a is observed in A549 cells. Live-cell image of CoV-2 Orf3a GFP (green) using a A549 doxycycline-inducible CoV-2 Orf3a GFP 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 Orf3a GFP 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.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Clinical Proteomics, Membrane, Stable Transfection, Patch Clamp

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Surface biotinylation experiments using Xenopus oocytes injected with SARS-CoV-1 (CoV-1) Orf3a 2x-STREP , SARS-CoV-2 (CoV-2) Orf3a 2x-STREP , or water demonstrates PM localization of Orf3a constructs used for two-electrode voltage clamp studies. Western blot detecting Orf3a 2x-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 Orf3a 2x-STREP mRNA injected (purple, n=7) Xenopus oocytes following protocol described in . Water injected I-V trace is the same trace as in . ( 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 Orf3a 2x-STREP or ( E ) CoV-2 Orf3a 2x-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 Orf3a 2x-STREP (purple, n=7), or CoV-2 Orf3a 2x-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 Orf3a SNAP (red) or CoV-1 Orf3a SNAP (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 Orf3a SNAP (red, n=11) and CoV-1 Orf3a SNAP (blue, n=9). Figure 2—figure supplement 3—source data 1. | Raw unedited western blots and figures with the uncropped blots for .

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Injection, Construct, Western Blot, Patch Clamp

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 Orf3a depicting dimeric architecture (dark and light pink) and key structural elements. ( C ) 2D topology of CoV-2 Orf3a. The region forming the cytosolic dimer interface is shown (yellow). ( D ) Inspection of the CoV-2 Orf3a TM region for a pore, depicted as the minimal radial distance from its center to the nearest van der Waals contact (HOLE program) . A region too narrow to conduct ions (white) and an aqueous vestibule (dark blue) are highlighted. ( E ) Radius of the pore (from D ) as a function of the distance along the ion pathway. Dashed lines indicate the minimal radius that would permit a dehydrated ion. Blue and white colors follow ( D ). ( F ) Two layers of polar residues (1 and 2, cyan and orange) identified in the TM region, with a zoom in of each region. ( G ) Basic residues located in the aqueous vestibule (purple) with zoom in of the region. ( H ) Cutaway of the CoV-2 Orf3a molecular surface to view the aqueous vestibule is colored according to the electrostatic potential (APBS program) . Coloring: blue, positive (+10 kT/e) and red, negative (–10 kT/e).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Cryo-EM Sample Prep

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: Text color denotes the program Relion 3.1 (green) or cryoSPARC v3.0 (dark blue) ( ; ; ). Details are described in the Methods . Low-resolution density for MSP1D1 is visible in maps of CoV-2 Orf3a but does not reach high-resolution (red circle).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 of particles. ( B, F ) Fourier Shell Correlation (FSC) curves of the final 3D reconstructions. The overall map resolutions are 3.0 Å ( B ) or 3.4 Å ( F ) at the FSC cutoff of 0.143 (dotted line). ( C, G ) Local map resolutions were estimated using Relion 3.0 (SARS-CoV-2 Orf3a LE/Lyso) or Relion 3.1 (SARS-CoV-2 Orf3a PM) and are colored as indicated . ( D, H ) Model validation. Comparison of the FSC curves between the model and half map 1 (FSC work ), model and half map 2 (FSC free ) and model and full map (FSC full ) . ( I ) Crosslinking of SARS-CoV-2 Orf3a from isolated HEK293 cellular membrane to assess its oligomeric state. A band of the approximate molecular weight of a dimer (2mer) appears with the addition of bismaleimidohexane (BMH). ( J ) Superposition of the SARS-CoV-2 Orf3a LE/Lyso MSP1D1 (pink) and PM (blue) structures. ( K ) Superposition of the SARS-CoV-2 Orf3a PM MSP1D1 (blue) and previously published SARS-CoV-2 Orf3a (yellow orange, PDB ID: 7KJR) structures.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Biomarker Discovery, Comparison, Isolation, Membrane, Molecular Weight

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 nanodisc (tan), SARS-CoV-2 Orf3a PM MSP1D1-containing nanodisc (pink), SARS-CoV-2 LE/Lyso Saposin A-containing nanodisc (dark purple), SARS-CoV-1 Orf3a LE/Lyso MSP1D1-containing nanodisc (green).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Cryo-EM Sample Prep

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 fenestrations between TM1 and TM3 of neighboring subunits (Lipid Site 1) and TM2 and TM3 of the same subunit (Lipid Site 2). A DOPE lipid is modeled into Lipid Site 1 (orange) and Lipid Site 2 (purple) in each view. ( B–C ) Cutaway view from the extracellular space to view lipids modeled into the density. Two DOPE lipids are modeled into ( B ) Lipid Site 1 (orange) or ( C ) Lipid Site 2 (purple) with lipid density depicted (blue mesh, contoured at 7σ). Lipid Sites 1 and 2 are likely not occupied simultaneously since the orientation of R122 (yellow) would sterically clash with DOPE in Lipid Site 2 (red dotted line). ( D–F ) CoV-2 Orf3a interacts with Saposin A. ( D ) Side view of CoV-2 Orf3a in LE/Lyso Saposin A nanodiscs highlighting two subunits (dark and light pink) and 6 molecules of Saposin A (cyan), with DOPE shown (purple). ( E ) Zoom in from the extracellular side to highlight the CoV-2 Orf3a and Saposin A interaction. CoV-2 Orf3a residues within 5 Å from Saposin A are shown (light pink) with DOPE (purple). ( F ) Zoom in from the extracellular side to highlight DOPE in Lipid Site 2 (purple). Note that residue R122 (light pink) is rotated 135° from the CoV-2 Orf3a LE/Lyso MSP1D1 structure (compare with ; see also for direct comparison) and occludes Lipid Site 1. ( G–I ) SARS-CoV-1 (CoV-1) Orf3a interacts with MSP1D1. ( G ) Side view of CoV-1 Orf3a in LE/Lyso MSP1D1 nanodiscs highlighting two subunits (dark and light green) and two molecules of MSP1D1 (light blue), with DOPE shown (orange). ( H ) Same view as to highlight the CoV-1 Orf3a and MSP1D1 interaction. CoV-1 Orf3a residues within 5 Å of MSP1D1 are shown (green), with DOPE (orange) depicted. ( I ) View as in to highlight DOPE in Lipid Site 1 (orange). Similar to , residue R122 (green) is positioned near and clashes with Lipid Site 2.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Residue, Comparison

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: Text color denotes that the program Relion 3.1 (green) or cryoSPARC v3.0 (dark blue) ( ; ; ). Details are described in the Methods . The initial map generated for iterative heterogeneous refinement (dotted box) displayed both higher-resolution density for CoV-2 Orf3a (gray, sharpened map) and lower-resolution Saposin A molecules surrounding SARS-CoV-2 Orf3a (gray, unsharpened map).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Generated

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 of particles. ( B, F ) Fourier Shell Correlation (FSC) curves of the final 3D reconstructions. The overall map resolutions are 2.8 Å ( B ) or 3.1 Å ( F ) at the FSC cutoff of 0.143 (dotted line). ( C, G ) Local map resolutions were estimated using Relion 3.1 and are colored as indicated . ( D, H ) Model validation. Comparison of the FSC curves between the model and half map 1 (FSC work ), model and half map 2 (FSC free ) and model and full map (FSC full ) . ( I ) Superposition of the SARS-CoV-2 Orf3a LE/Lyso MSP1D1 (pink) and SARS-CoV-2 Orf3a LE/Lyso Saposin A (purple) structures. ( J ) Superposition of the SARS-CoV-2 Orf3a (pink) and SARS-CoV-1 Orf3a (green) LE/Lyso MSP1D1 structures.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Biomarker Discovery, Comparison

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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, depicted as the minimal radial distance from its center to the nearest van der Waals protein contact (HOLE program) A region too narrow to conduct ions (white) and an aqueous vestibule (dark blue) are highlighted. ( B ) Radius of the ion pore (from A ) as a function of the distance along the ion pathway. Dashed lines indicate the minimal radius that would permit a dehydrated cation. Blue and white colors follow HOLE diagram of ( A ). ( C ) Cutaway of the CoV-1 Orf3a molecular surface to view the aqueous vestibule is colored according to the electrostatic potential (APBS program) . Coloring: blue, positive (+10 kT/e) and red, negative (–10 kT/e).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Schematic of the ACMA-based fluorescence flux assay ( ; ; ; ). 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 Na 2 SO 4 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) Orf3a 2x-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 Orf3a 2x-STREP -reconstituted proteoliposome patch with vesicle reconstituted at a 1:100 protein to lipid ratio. NaCl, n=27; KCl, n=32, and CaCl 2 , n=105. Error is represented as SEM.

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Fluorescence, Flux Assay, Generated, Control

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A–B ) K + ( A ) or Cl - ( B ) flux is not observed in SARS-CoV-2 (CoV-2) Orf3a 2x-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 Orf3a 2x-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 ( ; ). 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 Orf3a 2x-STREP reconstituted using 1:10 ( E , black) or 1:100 ( F , red) ratio of protein to lipids, and control vesicles ( G , blue).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Control, Flux Assay, Concentration Assay

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A ) Symmetrical recording solutions with CaCl 2 (green), KCl (blue) or NaCl (orange) used for proteoliposome patch-clamp experiments. ( B ) Multiple K + , Na + and Ca 2+ conductance species are observed by proteoliposome patch-clamp with SARS-CoV-2 (CoV-2) Orf3a 2x-STREP -containing vesicles reconstituted at a 1:10, but not 1:100 , ratio of protein to lipid. ( C ) Probability of observing an open event in a CoV-2 Orf3a 2x-STREP reconstituted proteoliposome patch with vesicles reconstituted at a 1:10 ratio of protein to lipid. NaCl, n=18; KCl, n=114; and CaCl 2 , 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 Orf3a 2x-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 Ca 2+ currents observed with proteoliposome patches-reconstituted CoV-2 Orf3a 2x-STREP , whereas DMSO has no effect (blue). ( F ) Open probability of proteoliposome patches-reconstituted CoV-2 Orf3a 2x-STREP sample following protocol from ( E ).

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Patch Clamp, Mass Spectrometry

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( A–B ) Rab7 puncta (green) are abundant in HEK293 cells expressing ( A ) SARS-CoV-2 (CoV-2) Orf3a HALO (magenta), but not ( B ) SARS-CoV-1 (CoV-1) Orf3a HALO (magenta; Hoechst 33342, blue). ( C ) Co-immunoprecipitation (co-IP) evaluating the interaction of VPS39 GFP with CoV-1 and CoV-2 Orf3a 2x-STREP , detected by western blot with antibodies against GFP and streptavidin, respectively. VPS39 GFP elutes with CoV-2 Orf3a 2x-STREP in a concentration-dependent manner, but does not elute with purified CoV-1 Orf3a 2x-STREP (compare VPS39 in d lanes, orange). Control, co-IP without Orf3a 2x-STREP added (bottom left, no protein). VPS39 GFP and Orf3a 2x-STREP migrate at ~130 and 35 kDa, respectively, by SDS-PAGE. ( D–I ) An unstructured loop of CoV-2 Orf3a partially mediates its interaction with VPS39. ( D ) Side view of CoV-2 Orf3a structure with the subunits (dark and light pink) and unstructured loop highlighted (yellow, dotted box). Zoom-in of the loop from the cytosol (solid box) with resolved loop residues. ( E ) CoV-2 Orf3a (red) and CoV-1 Orf3a (blue green) loop sequences. Orf3a wild-type (WT) and loop chimeras (LC) are color matched or swapped. Created with Biorender.com ( F ) Co-IP as in with CoV-2 Orf3a constructs showing loss of VPS39 GFP elution with CoV-2 Orf3a LC 2x-STREP (compare VPS39 in d lanes, orange). The co-IPs presented in this figure represent three to seven independent experiments. ( G ) Co-IP of VPS39 GFP with CoV-1 Orf3a constructs shows an enrichment with CoV-1 Orf3a LC 2x-STREP . ( H, I ) Rab7 puncta (green) are absent in CoV-2 Orf3a LC HALO ( H , magenta) or CoV-1 Orf3a LC HALO -expressing HEK293 cells ( I , magenta; Hoechst 33342, blue), consistent with . ( J–K ) Cumming estimation plots of Rab7 puncta from ( A, H ) ( J ) and ( B, I ) ( K ) . Figure 6—source data 1. Raw unedited western blots and figures with the uncropped blots for . Figure 6—source data 2. Raw unedited western blots and figures with the uncropped blots for .

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Concentration Assay, Purification, Control, SDS Page, Construct

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 fraction is highlighted in gray. ( B, D ) Gel filtration traces of final samples of ( B ) CoV-2 Orf3a LC and ( D ) CoV-1 Orf3a LC used for the co-IP experiments .

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Filtration, Co-Immunoprecipitation Assay

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: ( 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 between SARS-CoV-2 Orf3a and VPS39 . Putative VPS39 interfaces are indicated (light blue spheres) with potential stoichiometries of 1:1 or 1:2 molecules of VPS39 to SARS-CoV-2 Orf3a. ( B ) Cytosolic surface of SARS-CoV-2 and SARS-CoV-1 Orf3a colored by their electrostatic potential (APBS program): blue, positive (+5 kT/e); red, negative (–5 kT/e) . The putative VPS39 interfaces (dotted black lines) are the same as indicated in ( A ). ( C ) Working model of SARS-CoV-2 Orf3a dysregulation of late endosome and autophagosome fusion with lysosomes. HOPS-dependent regions of the endocytic and autophagy pathways that are disrupted by SARS-CoV-2 Orf3a are indicated (red X). Adapted from “Mutation of HOPS Complex Subunits”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates .

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Mutagenesis

Journal: eLife

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

doi: 10.7554/eLife.84477

Figure Lengend Snippet: Cryo-EM data processing workflow for SARS-CoV-2 Orf3a reconstituted in LE/Lysosomal MSP1D1-containing nanodiscs, continued from .

Article Snippet: For protein expression, SARS-CoV-1 Orf3a or SARS-CoV-2 Orf3a P2 virus (3–10% of total cell suspension volume) was used to infect HEK293S GnTI- cells, grown in FreeStyle 293 Expression Medium (Thermo Fisher) adapted to suspension growth in 2% Foundation FBS (Gemini Bio Products).

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex.

doi: 10.1016/j.cell.2022.09.038

Figure Lengend Snippet: Figure 1. MAPseq and BARseq projection mapping of individual olfactory bulb neurons (A) Schematics of the MAPseq strategy which uses RNA barcodes to label neurons and map their brain-wide projections. (B) Infection of mitral and tufted cells by Sindbis virus carrying the barcodes and a fluorophore (EGFP). (C) Laser Capture Micro-Dissection of target brain regions from Nissl stained coronal sections registered to the Allen Brain reference atlas. (D) Illustration of laminar positions of mitral, tufted, and deep cells (left) and an example BARseq sequencing image of the barcoded cells (center). The first several bases of the barcode sequences in two example neurons analyzed via BARseq and their projection patterns across 6 bulb target regions (right). Scale bar = 100mm. (E) (Left) projection patterns (415 neurons, 2 mice) identified via BARseq and their soma locations relative to the mitral cell layer (MCL). Columns represent bulb projection target regions and rows indicate individual neurons. (right) Cell identities based on soma positions. Projection strength of each barcoded neuron has been normalized so that the maximum strength is 1 (row). (F) (Left) Soma positions of template neurons shown relative to MCL (y axis) and to glomerular layer (x axis) that were used to train the projection-based classifier. The sectioning planes are not necessarily perpendicular to the mitral cell layer, and thus the distances measured may be inflated. Neuronal identity (colors) is based on laminar positions (tufted, mitral, and deep cells). (Right) Classification confusion matrix of all three cell classes using the BARseq-based classifier versus position-defined classes. See also Figures S1 and S2.

Article Snippet: Barcoded Sindbis viral library Abarcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used forMAPseq andBARseq experiments as described previously (Chen et al., 2019; Han et al., 2018; Huang et al., 2020; Kebschull et al., 2016).

Techniques: Infection, Virus, Dissection, Staining, Sequencing

Journal: bioRxiv

Article Title: Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

doi: 10.1101/2021.05.12.443929

Figure Lengend Snippet: ( a ) Schematics of the MAPseq strategy which uses RNA barcodes to label neurons and map their brain-wide projections. ( b ) Infection of mitral and tufted cells by Sindbis virus carrying the barcodes and a fluorophore (GFP). ( c ) Laser Capture Micro-Dissection of target brain regions from Nissl stained coronal sections and corresponding sections registered to the Allen Brain reference atlas. ( d ) Illustration of laminar positions of mitral, tufted, and deep cells (Left) and an example BARseq sequencing image of the barcoded cells (Center). The first several bases of the barcode sequences in two example neurons analyzed via BARseq and their projection patterns across 6 bulb target brain regions (Right). Scale bar = 100 µm. ( e ) Projection patterns of neurons (415 neurons, 2 mice) identified via BARseq and their soma locations relative to the mitral cell layer (MCL). Columns represent olfactory bulb projection target regions and rows indicate individual neurons. Cell identities based on soma positions are shown on the right. Projection strength of each barcoded neuron has been normalized so that the maximum strength is 1 in each neuron (row). ( f ) (Left) Soma positions of template neurons shown relative to MCL (y-axis) and to glomerular layer (x-axis) that were used to train the projection-based classifier. Neurons are colored by their identities based on laminar positions (tufted, mitral and deep cells). (Right) The classification confusion matrix of all three classes of neurons using the BARseq-based classifier versus the position-defined classes. ( g )-( i ) The projection patterns of all MAPseq analyzed neurons ( g ), their mean projection patterns ( h ), and five example neurons ( i ) of the three classes of bulb projection neurons identified via a BARseq-based classifier. In ( g ), columns represent projection brain regions and rows indicate individual barcoded neurons. Barcoded neurons are sorted by probability of cell type classification based on running their projection patterns through the classifier. ( j ) Distribution of the broadness of projections, as measured by Inverse Participation Ratio (IPR, x-axis) at brain region-level. ( k ) Pearson correlation between putative mitral cell (pMC) projections to different target regions. Only correlations that passed statistical significance after Bonferroni correction are shown. ( l ) Distribution of the city block distance between the projection patterns of each pMC identified using the BARseq-based classifier and the most similarly projecting pMC within the same brain (blue), across different brains (red), across all brains (6) sampled after shuffling all elements in the projection matrix (yellow), or after shuffling the neuron identities for each area separately (purple).

Article Snippet: A barcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used for MAPseq and BARseq experiments as described previously – .

Techniques: Infection, Virus, Dissection, Staining, Sequencing, Blocking Assay

Journal: bioRxiv

Article Title: Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

doi: 10.1101/2021.05.12.443929

Figure Lengend Snippet: ( a ) (Left) Projection patterns of piriform cortex output neurons to extra-piriform brain regions (Supplementary Table 4) and (Right) within the piriform cortex along the A-P axis. Projection density is color-coded on a log scale. In the piriform cortex, for a given barcoded neuron, the A-P position with the most barcode counts is taken as the location of the soma. ( b ) Mean projection strengths (log scale) of projections from somata at the indicated locations (x-axis) to the specific A-P positions within the piriform cortex (y-axis). ( c ) Differences in reciprocal projections between two A-P positions in the piriform cortex obtained by calculating the difference between the connectivity matrix (b) and its transpose. Blue indicates stronger projection in the posterior direction, and red indicates stronger projection in the anterior direction. ( d ) (Left) The strength of intra-piriform projections relative to their soma locations (blue). Red line indicates fit using an inverse power law model (Methods). The density of projections decreases by half at about 0.5 mm from soma location (maximum density of barcodes), making the projections width equal to 1 mm at 50% density (arrows). Contribution from dendritic neuropil of barcoded neurons was minimized by removing slices adjacent to the peak of barcode molecule counts (Methods). (Right) the same distribution obtained for pMC is substantially broader. ( e ) Mean projection patterns (Top) and the projection patterns of individual neurons (Bottom) of groups of piriform cortex output neurons to extra-piriform target brain regions. ( f ) Fraction of neurons belonging to each group at the indicated A-P positions within the piriform cortex. The color codes used are the same as in ( e ).

Article Snippet: A barcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used for MAPseq and BARseq experiments as described previously – .

Techniques:

Journal: bioRxiv

Article Title: Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

doi: 10.1101/2021.05.12.443929

Figure Lengend Snippet: ( a ) Mean projection patterns of piriform cortex output neurons at the indicated A-P position of barcoded somata in the piriform cortex. Dotted lines indicate linear fits and shaded areas the range of fits from bootstrap. ( b ) Mean loadings for the first two principal components of the mean projection strengths of piriform output neurons to AON, CoA, lENT and OT sampled at the indicated A-P positions in the piriform cortex. Dotted lines indicate linear fits for APC and PPC. ( c ) Mean projection strengths of piriform projection neurons to extra-piriform target regions, organized by the location of their somata along the A-P axis of the piriform cortex (y-axis) plotted against the mean projection strengths of pMC neurons to extra-piriform bulb target regions weighted by projections to a particular A-P position in piriform cortex, P(target|PC location) (x-axis). Colors indicate A-P positions in the piriform cortex. ( d ) Cartoon schematics of the parallel olfactory circuits engaging the olfactory bulb-to-piriform inputs, piriform cortex (APC and PPC) outputs and extra-piriform bulb target regions (AON, CoA and lENT) sampled in this study.

Article Snippet: A barcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used for MAPseq and BARseq experiments as described previously – .

Techniques: