Journal: Molecular Brain

Article Title: Development of a novel peptide to prevent entry of SARS-CoV-2 into lung and olfactory bulb cells of hACE2 expressing mice

doi: 10.1186/s13041-022-00956-1

Figure Lengend Snippet: Peptides are able to block the interaction between ACE2 and SARS-CoV-2 spike protein, or inhibit the cleavage of spike protein by protease. A Schematic diagram of design strategy for peptides to block SARS-CoV-2 virus from entering cells. B Representative blot image showing that ACE2 can be co-immunoprecipitated by anti-Spike protein (S protein) antibody, but not IgG, in HEK293T cells transfected with S protein and hACE2. C Densitometric analysis of the levels of ACE2 co-immunoprecipitated by anti-S protein antibody in HEK293T cells transfected with S protein and ACE2 shows that TAT-ACE2-1, TAT-ACE2-2 and TAT-ACE2-3 peptides, but not TAT alone, are able to decrease the level of S-ACE2 complex. The level of co-immunoprecipitated ACE2 (ACE2 Co-IP) was normalized to the level of precipitated S protein (S protein IP). Data are shown as mean ± SEM, and presented as the percentage of the control (vehicle) sample. n = 3 independent experiments for each group, one-way ANOVA test followed by Dunnett’s post hoc test. *p < 0.05, **p < 0.01 as compared to control (vehicle) samples. D Western blot shows that TAT-S1/S2 or TAT-S2 peptides are able to inhibit S protein cleavage by TMPRSS2 in HEK293T cells transfected with S protein, ACE2 and TMPRSS2. α-Tubulin was used as a loading control. E Densitometric analysis of the levels of S2 in HEK293T cells transfected with S protein, ACE2 and TMPRSS2, and treated with either vehicle (control), TAT alone, TAT-S1/S2 or TAT-S2 peptides. The level of S2 was normalized to the level of α-Tubulin. Data are shown as mean ± SEM, and presented as the percentage of the control (vehicle) sample. n = 3 independent experiments for each group, one-way ANOVA test followed by Dunnett’s post hoc test. *p < 0.05 as compared to control (vehicle) samples

Article Snippet: The plasmids encoding the SARS-CoV-2 spike protein (S protein) (pTwist-EF1 alpha-SARS-CoV-2-S-2xStrep was a gift from Nevan Krogan [ ] (Addgene plasmid #141,382)), human ACE2 (hACE2 was a gift from Hyeryun Choe [ ] (Addgene plasmid #1786; http://n2t.net/addgene:1786 ; RRID: Addgene_1786)), and TMPRSS2 (TMPRSS2 was a gift from Roger Reeves [ ] (Addgene plasmid #53,887; http://n2t.net/addgene:53887 ; RRID: Addgene_53887)) were purchased from Addgene.

Techniques: Blocking Assay, Virus, Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, Control, Western Blot

Journal: Molecular Brain

Article Title: Development of a novel peptide to prevent entry of SARS-CoV-2 into lung and olfactory bulb cells of hACE2 expressing mice

doi: 10.1186/s13041-022-00956-1

Figure Lengend Snippet: TAT-peptides inhibit SARS-CoV-2 S protein-mediated cell–cell fusion in HEK293T cells. A Sample microscopic images of SARS-CoV-2 S protein-mediated cell–cell fusion in HEK293T cells treated with either vehicle (Control), ACE2 antibody (2 µg/ml, 1 h), TAT alone, TAT-ACE2-1, TAT-ACE2-2, TAT-ACE2-3, TAT-S1/S2, or TAT-S2 peptides at 10 µM (scale bar: 100 µm). White arrows indicate the fused cells. B TAT-ACE2-1, TAT-ACE2-2, TAT-ACE2-3, TAT-S1/S2, TAT-S2, or ACE2 antibody each inhibit SARS-CoV-2 S protein-mediated cell–cell fusion. The number of fused cells was expressed as a fraction of total cell number in the same image. Data are shown as mean ± SEM, and presented as the percentage of the control (vehicle) samples. n = 10 – 21 images for each group, one-way ANOVA test followed by Dunnett’s post hoc test. ****p < 0.0001 as compared to control (vehicle) samples

Article Snippet: The plasmids encoding the SARS-CoV-2 spike protein (S protein) (pTwist-EF1 alpha-SARS-CoV-2-S-2xStrep was a gift from Nevan Krogan [ ] (Addgene plasmid #141,382)), human ACE2 (hACE2 was a gift from Hyeryun Choe [ ] (Addgene plasmid #1786; http://n2t.net/addgene:1786 ; RRID: Addgene_1786)), and TMPRSS2 (TMPRSS2 was a gift from Roger Reeves [ ] (Addgene plasmid #53,887; http://n2t.net/addgene:53887 ; RRID: Addgene_53887)) were purchased from Addgene.

Techniques: Control

Journal: Molecular Brain

Article Title: Development of a novel peptide to prevent entry of SARS-CoV-2 into lung and olfactory bulb cells of hACE2 expressing mice

doi: 10.1186/s13041-022-00956-1

Figure Lengend Snippet: TAT-peptides inhibit SARS-CoV-2 S protein entry into HEK293T cells. A Representative fluorescent microscopy images are shown. HEK293T cells transiently transfected with hACE2 and TMPRSS2 were pre-incubated with vehicle, ACE2 antibody, TAT alone or related TAT-peptides (10 µM), and inoculated with pseudotyped VSV expressing SARS-CoV-2 S protein. Pseudotyped VSV contains a GFP reporter gene. ACE2 antibody was used as a positive control. Scale bar: 100 µm. B TAT-ACE2-1, TAT-ACE2-2, TAT-ACE2-3, TAT-S1/S2, or TAT-S2 inhibit the SARS-CoV-2 S protein entry into HEK293T cells. The fluorescent intensity of GFP in live cells was quantified using a Synergy H4 plate luminometer. Data are shown as mean ± SEM, and presented as the percentage of the control (vehicle) samples. n = 8 – 14 samples for each group, one-way ANOVA test followed by Dunnett’s post hoc test. *p < 0.05, ***p < 0.001, ****p < 0.0001 as compared to control (vehicle) samples. #p < 0.05, ###p < 0.001 as compared to TAT-ACE2-2 samples. C Analysis of EC50 of TAT-ACE2-2 peptide for inhibiting VSV-ΔG-GFP-SARS-CoV-2 pseudovirus entry into HEK 293 T cells transfected with hACE2 and TMPRSS2. Eight dilutions ranging from 3 nM to 3 µM were assessed. D TAT-ACE2-2 inhibits the binding of purified S protein and ACE2. The RBD domain of SARS-CoV-2 S protein was coated onto a 96-well plate, and the same concentration of purified ACE2 together with various concentrations of TAT-ACE2-2 were simultaneously added into each well. ACE2 binding to S protein was detected using anti-ACE2 antibody and HRP-conjugated secondary antibody; TMB one-step substrate reagent was added to visualize HRP. Data are shown as mean ± SEM, and presented as the percentage of the control samples (no TAT-ACE2-2 added, 0 mM). n = 3 samples for each group; one-way ANOVA test followed by Dunnett’s post hoc test. *p < 0.05, ****p < 0.0001 as compared to control (0 mM) samples

Article Snippet: The plasmids encoding the SARS-CoV-2 spike protein (S protein) (pTwist-EF1 alpha-SARS-CoV-2-S-2xStrep was a gift from Nevan Krogan [ ] (Addgene plasmid #141,382)), human ACE2 (hACE2 was a gift from Hyeryun Choe [ ] (Addgene plasmid #1786; http://n2t.net/addgene:1786 ; RRID: Addgene_1786)), and TMPRSS2 (TMPRSS2 was a gift from Roger Reeves [ ] (Addgene plasmid #53,887; http://n2t.net/addgene:53887 ; RRID: Addgene_53887)) were purchased from Addgene.

Techniques: Microscopy, Transfection, Incubation, Expressing, Positive Control, Control, Binding Assay, Purification, Concentration Assay

Journal: Journal of Clinical and Translational Science

Article Title: Detecting SARS-CoV-2 Orf3a and E ion channel activity in COVID-19 blood samples

doi: 10.1017/cts.2021.856

Figure Lengend Snippet: Orf3a channel protein sequence alignment in SARS-CoV-2, SARS-CoV, bat SARS-CoV, HCoV-229E, and HCoV-NL63 coronaviruses. Red arrows label three amino acid residues critical for K+ permeation of the channel.

Article Snippet: Lentivirus containing SARS-CoV-2 Orf3a and lentivirus containing E were obtained from Addgene donated by Nevan Krogen lab: pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro was a gift from Nevan Krogan (viral prep # 141383-LV; http://n2t.net/addgene:141383-LV ; RRID:Addgene_141383) [ ]. pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro was a gift from Nevan Krogan (viral prep # 141385-LV; http://n2t.net/addgene:141385-LV; RRID:Addgene_141385) [ ].

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: Sequences of the C-terminal tails of the ORF6 and hIFN γ proteins.

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: Number of H-bonds between ( a ) ORF6 and the CT-hIFN γ peptides; and ( b ) hIFN γ and the ORF6 proteins.

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: Maps of the close contacts between ( a ) ORF6 and the CT-hIFN γ peptides; and ( b ) hIFN γ and the ORF6 proteins. A close contact is considered present if two heavy atoms from the two interacting molecules are within 4.5 Å of each other.

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: Binding of hIFN γ and three ORF6 proteins. hIFN γ is depicted in dark gray, ORF6-1 to ORF6-4 are shown, respectively, in red, blue, green, and pink cartoons. The ER membrane is a light gray surface representation.

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques: Binding Assay, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: RAE1 localisation in ORF6 overexpressing cells upon hIFN γ treatment. ( a ) Non-treated and hIFN γ -treated cells, overexpressing ORF6, were stained with antibodies against RAE1 and ORF6. Representative images are shown. Scale bar is 5 μ m. ( b ) The fluorescence intensity of RAE1 (in the green channel) was analysed for each cell using the ImageJ version 1.54h. software in ORF6 oe cells and ORF6 oe cells upon hIFN γ treatment. The difference is statistically significant with a **** p -value < 0.0001, estimated using Student’s test for at least three independent experiments (two-tailed unpaired Student’s test).

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques: Staining, Fluorescence, Software, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: Levels of expression of RAE1 in ORF6 overexpressing cells upon hIFN γ treatment. Control–cells transfected with empty vector; control + hIFN γ –cells transfected with empty vector and treated with hIFN γ ; ORF6 oe–cells transfected with plasmid coding for ORF6; ORF6 oe + hIFN γ –cells transfected with plasmid coding for ORF6 and treated with hIFN γ . The figures shown are based on at least three independent experiments and are represented as mean ± standard error of the mean (error bars), **** p -value < 0.0001, ** p -value < 0.01, estimated using Student’s test for at least three independent experiments (two-tailed unpaired Student’s test).

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques: Expressing, Control, Transfection, Plasmid Preparation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: GFP reporter expression. ( a ) Visualisation of GFP expression under fluorescence microscope. Representative images are shown. Scale bar is 100 μ m. ( b ) The fluorescence intensity of GFP was analysed using ImageJ software. ( c ) qRT-PCR of the nuclear levels of GFP mRNA isolated from ( a ). Control–cells transfected with GFP; hIFN γ treatment–cells transfected with GFP and treated with hIFN γ ; ORF6 oe–cells co-transfected with plasmids coding for ORF6 and GFP; ORF6 oe + hIFN γ –cells co-transfected with plasmids coding for ORF6 and GFP and treated with hIFN γ . The results are based on at least three independent experiments and are represented as mean ± standard error of the mean (error bars), **** p -value < 0.0001, ** p -value < 0.01, based on at least three independent experiments (two-tailed unpaired Student’s t -test).

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques: Expressing, Fluorescence, Microscopy, Software, Quantitative RT-PCR, Isolation, Control, Transfection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Interferon- γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

doi: 10.3390/ijms25042155

Figure Lengend Snippet: hIFN γ inhibits the formation of R-loops and reinstates the rates of replication fork progression. ( a ) Replication rates. **** p -value < 0.0001, ns—not significant ( p -value > 0.05), based on at least three independent experiments (two-tailed unpaired Student’s t -test). ( b ) FRAP analysis of RBD-DsRed in control, ORF6-overexpressing cells, and ORF6-overexpressing cells, treated with hIFN γ . Control–cells, transfected with empty vector; control + hIFN γ –cells transfected with empty vector and treated with hIFN γ ; ORF6 oe–cells transfected with plasmid coding for ORF6; ORF6 oe + hIFN γ –cells transfected with plasmid coding for ORF6 and treated with hIFN γ .

Article Snippet: The plasmid pLVX-EF1alpha-SARS-CoV-2-orf6-2xStrep-IRES-Puro (Addgene plasmid #141387 from Nevan Krogan) [ ] was used for the overexpression of the accessory proteins ORF6.

Techniques: Two Tailed Test, Control, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: SARS-CoV-2 ORF8 is an ER luminal protein. A . Amino acid sequence analysis (Protter) of ORF8 and prediction as an ER luminal protein (due to the presence of ER signal peptide and the absence of a transmembrane domain). B . A549 cells transfected with a plasmid encoding ORF8-Strep were fixed, permeabilized, and immunostained for Strep (ORF8) or PDI (ER marker), which were analyzed by fluorescence confocal microscopy imaging. The cells were counterstained using DAPI and Phalloidin (upper left). The dashed box is digitally enlarged to show colocalization (bottom right) of ORF8 (upper right) and ER (bottom left). White scale bars = 10 μm. C . The pixel intensities of ORF8 and PDI along the dashed arrow in B are plotted. D . HEK293T cells transfected with a plasmid encoding ORF8-Flag were mechanically lysed and fractionated by differential centrifugation. The indicated subcellular fractions were evaluated by immunoblot analysis for Flag (ORF8), Calnexin (ER marker), COX4 (mitochondrial marker) or β-actin (cytosolic marker). E . The ER subcellular fractions in D were further incubated with two concentrations of digitonin (0, 0.035, or 0.2%). The fractions were then centrifuged, and the supernatants containing digitonin-solubilized proteins were evaluated by immunoblot analysis as in D . B – E . The data represent three independent experiments.

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Sequencing, Transfection, Plasmid Preparation, Marker, Fluorescence, Confocal Microscopy, Imaging, Centrifugation, Western Blot, Incubation

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: ORF8 colocalizes with Spike and modulates Spike protein levels and furin-dependent processing. A . A549 cells co-transfected with plasmids encoding Spike-Flag or ORF8-Strep were fixed, permeabilized, and immunostained for Flag (Spike) and Strep (ORF8), which were analyzed by fluorescence confocal microscopy imaging. The cells were counterstained using DAPI and Phalloidin, (upper left), with ORF8 (upper right), Spike (bottom left), or both ORF8 and Spike signals merged (bottom right). White scale bars = 10 μm. B , C , E – H . HEK293T cells co-transfected with plasmids encoding Spike-Flag ( B , C , E – G ) or SARS-CoV-derived Spike ( H ), or, eGFP-Strep ( B , C ), ORF8-Strep ( B , C , E – H ), ORF8-Strep S84L (the B lineage genotype) ( E , F ), or SARS-CoV-derived ORF8-Strep genotypes (ORF8ab, ORF8a, ORF8b, or ORF8a and ORF8b together) ( G , H ) were lysed for immunoblot analysis using antibodies against S2 (detects uncleaved and S2 fragment of Spike, or SARS-CoV Spike) ( B , C , E – H ), S1 fragment (detects uncleaved and S1 fragment of Spike) ( B , C ), Strep (detects eGFP or ORF8s) ( B , C , E – H ), and β-actin ( B , C , E – H ). D . Depiction of whole Spike with the site that can be cleaved by furin, yielding S1 and S2 fragments. Apparent immunoblot mass (kDa) is indicated. The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed by one-way ANOVA (Dunnett’s test).

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Transfection, Fluorescence, Confocal Microscopy, Imaging, Derivative Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: ORF8 covalently interacts with Spike and hampers Spike translocation to the Golgi apparatus. A , D – G . HEK293T cells co-transfected with plasmids encoding non-tagged Spike ( A , D , F , G ), or Spike-Strep ( E ), and/or GFP-Flag ( A ), ORF8-Flag ( A , E , F , G ), ORF8-Flag I9P (non-ER mutant) ( A ), or ORF8-Flag Δ1-17 (ER signal deletion) ( A ) were not infected ( A , D – G ) or infected ( D ) with VSVΔ G-GFP, for production of a hybrid VSVΔG-GFP that incorporated fully mature SARS-CoV-2 Spike (S-VSV). The cells above expressing Spike and/or ORF8 constructs, or, S-VSV collected from the culture medium were lysed and directly analyzed by immunoblots using antibodies against S2 (detects uncleaved and S2 fragment of Spike) ( A , D , F ), N-terminus of S2 (detects S2 fragment only) ( A , D , F ), Flag (detects GFP or ORF8) ( A , E , F , G ), Strep (detects Spike) ( E ), and β-actin, under reducing or non-reducing (protein interactions through disulfide bonds were preserved) conditions or further incubated with anti-Flag magnetic beads ( A , F , G ), without ( A , F , G ) or with pre-treatment ( F , G ) (denaturation; 2% SDS, 5 min at 95 °C, reduction: 0.02% β-ME) of the cell lysates. The proteins that were immunoprecipitated were analyzed by immunoblots under reducing ( A , F , G ) or non-reducing condition ( F , G ). B , C . A549 ( B ) or HEK293T ( C ) cells co-transfected with a plasmid encoding Spike-Flag and a bicistronic plasmid encoding ORF8 and eGFP (ORF8-Strep- IRES -eGFP) were fixed, permeabilized, and immunostained using antibodies against S2 (detects uncleaved and S2 fragment of Spike) and TGN46 (Golgi marker), which were analyzed by fluorescence confocal microscopy imaging. B . The cells that were not expressing or expressing ORF8-Strep (identified by eGFP signals, pseudo-colored to white) were counterstained using DAPI (upper left), with the dashed box that is digitally enlarged to evaluate colocalization (bottom right) of Spike (upper right) and Golgi (TGN46) (bottom left). White scale bars = 2 μm. C . Colocalization of Spike and Golgi signals within the circular area encompassing Golgi (dashed circles in B ) was analyzed by measuring Pearson’s coefficient in 60 cells (combined from three independent experiments, in which 20 cells were randomly selected). The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed using two-tailed Student’s t test ( C ) or one-way ANOVA (Dunnett’s test) ( G ).

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Translocation Assay, Transfection, Mutagenesis, Infection, Expressing, Construct, Western Blot, Incubation, Magnetic Beads, Immunoprecipitation, Plasmid Preparation, Marker, Fluorescence, Confocal Microscopy, Imaging, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: Host protein synthesis is inhibited in cells expressing ORF8. A – D . HEK293T cells co-transfected with a plasmid encoding Spike-Flag ( A , D ) or Spike-Flag FKO (furin cleavage site KO) ( B , C ), and eGFP-Strep ( B , C ) or ORF8-Strep ( A – D ), were lysed after incubation without ( B – D ) or with ( A ) CMK (furin inhibitor), and evaluated by immunoblot analysis ( A – C ) using antibodies against S2 (detects uncleaved and S2 fragment of Spike), Strep (detects GFP or ORF8), or β-actin, or, evaluated by RT-qPCR ( D ) using primers that are designed against Spike. E – I . HEK293T cells transfected with a bicistronic plasmid encoding both eGFP and different ORF8-Strep genotypes (ORF8-Strep, ORF8-Strep S84L, or, SARS-CoV-derived ORF8ab-Strep, ORF8a-Strep, or ORF8b-Strep) were incubated in the absence ( E , H ) or presence ( F , G , I ) of HPG (methionine analog) without or with puromycin (protein synthesis inhibitor) ( F – H ). After 30 min, the cells were harvested, fixed, and directly analyzed by flow cytometry for the fluorescence signals of eGFP ( E ), or permeabilized after fixation and fluorescently labeled for flow cytometry analysis of the incorporated cellular HPG within cells expressing ORF8-Strep (eGFP-positive) ( F , G , I ), or stained with LIVE/DEAD dye for evaluating the percentage of dead cells (LIVE/DEAAD-staining positive) using flow cytometry ( H ). The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed using one-way ANOVA ( C , E , G , H ; Dunnett’s test, I ; Tukey’s test) or two-tailed Student’s t test ( D ).

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Quantitative RT-PCR, Derivative Assay, Flow Cytometry, Fluorescence, Labeling, Staining, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: ORF8 limits the levels of cell-surface Spike. A . A monolayer of HEK293T cells stably expressing ACE2/TMPRSS2 were co-transfected with a plasmid encoding Spike-Flag or ORF8-Strep. The cells were fixed, permeabilized, and immunostained with antibodies against Flag (Spike) and Strep (ORF8). After counterstaining with DAPI, the cells were analyzed using fluorescence confocal microscopy imaging. The dotted boxes were digitally enlarged (upper right inset). The scale bars = 50 μm. B – H . HEK293T cells co-transfected with a plasmid encoding Spike-Flag (C-terminal tagged) ( B , C , E ), Flag-Spike-Flag (N- and C-terminal tagged) ( D , E ), non-tagged Spike ( F , G ), or SARS-CoV-derived Spike ( F – H ), and a bicistronic plasmid encoding eGFP and different ORF8-Strep genotypes (ORF8-Strep ( B – E , H ), ORF8-Strep S84L ( B , C ) or SARS-CoV-derived ORF8ab-Strep, ORF8a-Strep, or ORF8b-Strep ( H )). The cells were harvested and directly immunostained for cell surface Spike by incubating with antibodies against S2 ( B – D , G , H ; detects uncleaved Spike, S2 fragment, or SARS-CoV-derived Spike) or Flag ( E ) or fixed, permeabilized and immunostained by incubating with antibodies against S2 ( G ). Cell-surface Spike levels ( B – H ) in viable (LIVE/DEAD-negative) cells expressing ORF8-Strep (eGFP-positive) or total cellular Spike levels ( G ) in cells expressing ORF8-Strep (eGFP-positive) were measured by flow cytometry. Cellular expression levels of SARS-CoV-2 Spike or SARS-CoV Spike were confirmed by immunoblot analysis ( F ). The data represent or are combined from three independent experiments and presented as mean ± s.d. Statistical significance was analyzed using one-way ANOVA ( C , E , H ; Dunnett’s test) or two-tailed Student’s t test ( D , G ).

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy, Imaging, Derivative Assay, Flow Cytometry, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: The ORF8 cellular effects are dependent on the ORF8 expression levels, which were detected lower than ORF8 expression levels in SARS-CoV-2 infection. A , B . A monolayer of HEK293T cells were co-transfected with a bicistronic eGFP plasmid encoding none (top) or ORF8-Flag (middle) without ( A ) or with ( B ) a plasmid encoding Spike-Flag. After 30 min incubation in the absence ( B ) or presence ( A ) of HPG (methionine analog that can be fluorescently labeled), the cells were fixed, permeabilized, and fluorescently labeled for incorporated HPG ( A ), or immunostained for cell surface Spike levels using antibodies against Spike S2 ( B ). The cells were analyzed by flow cytometry and gated into 4 groups based on the transfection levels (eGFP) (10 0.5 fold increment). C , D . HEK293T cells stably expressing ACE2/TMPRSS2 were transfected with a bicistronic eGFP plasmid encoding ORF8-Strep for 18 h, or infected with SARS-CoV-2 at the MOI of 0.1 for 48 h. The cells were lysed, and analyzed by RT-qPCR using primers targeting ORF8 ( C ), or directly evaluated by flow cytometry analysis for relative ORF8 expression levels (eGFP signals) ( D ). The average eGFP signals of the whole transfected population is indicated (dotted line within green gradient), which was overlayed by the total ORF8 expression levels in the whole cell populations detected in C (gradient indicates s.d.). The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed using two-tailed Student’s t test.

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Expressing, Infection, Transfection, Plasmid Preparation, Incubation, Labeling, Flow Cytometry, Stable Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: ORF8 limits the reactivity of anti-SARS-CoV-2 (convalescent or vaccinated) human sera towards Spike-producing cells. A – C . HEK293T cells co-transfected with a plasmid encoding Spike-Flag and a bicistronic plasmid encoding mCherry and ORF8-Strep genotypes (ORF8-Strep, ORF8-Strep S84L) were harvested and incubated with anti-SARS-CoV-2 human sera collected from three COVID-19 convalescent ( A , B ), six vaccinated ( C ) (three Pfizer and three Moderna, before 1st shot (pre-vaccination) and after 2nd shot (post-vaccination)), or three COVID-19-negative donors ( A ). The IgG molecules in the sera that reacted to Spike-producing cells were fluorescently labeled using antibodies that specifically recognize the Fc region of human IgG molecules. The human IgG Fc levels in viable (LIVE/DEAD-negative) cells expressing ORF8-Strep (mCherry-positive) were evaluated by flow cytometry. The data are combined from three independent experiments and represented as mean ± s.d. Statistical significance was analyzed using one-way ( B , Dunnett’s test) or two-way ANOVA ( C , Tukey’s test).

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Transfection, Plasmid Preparation, Incubation, Labeling, Expressing, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells

doi: 10.1016/j.jbc.2023.104955

Figure Lengend Snippet: ORF8 results in decreased infectivity of Spike-pseudotyped virus (S-VSV), but limits the reactivity of anti-SARS-CoV-2 human sera towards the infected cells. A . Experimental workflow and summary of results. B . HEK293T cells co-transfected with plasmids encoding Spike or ORF8-Strep genotypes (ORF8-Strep, or ORF8-Strep S84L) were infected with replication-incompetent GFP-encoding VSV (VSVΔG-GFP), which resulted in production of Spike incorporated ΔG-GFP (S-VSV). The culture medium containing S-VSV was collected, and Spike levels in S-VSV were evaluated by immunoblot analysis with antibodies against S2 (detects both uncleaved and S2 fragment) and VSV-M (loading control, a VSV structural protein). C – F . HEK293T cells stably expressing ACE2/TMPRSS2 were infected with S-VSV produced in the absence or presence of ORF8-Strep or ORF8-Strep S84L. C . The infectivity of S-VSVs were evaluated by flow cytometry analysis for the percentage of GFP expressing cells. The cells were further incubated with antibodies against S2 (detects both uncleaved and S2 fragment) ( D ), or anti-SARS-CoV-2 human sera collected from three COVID-19 convalescent ( E ), or six vaccinated ( F ) (three Pfizer and three Moderna, before 1st shot (pre-vaccination) and after 2nd shot (post-vaccination)) donors. The levels of cell-surface Spike in S-VSV infected cells ( D ) and the reactivity of anti-SARS-CoV-2 human sera ( E and F ) towards S-VSV-infected cells were evaluated flow cytometry in S-VSV-infected (GFP-positive), but yet viable (LIVE/DEAD-negative) cells. The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed using one-way ( B – E ; Dunnett’s) or two-way ( F ; Tukey’s) ANOVA.

Article Snippet: Several plasmids were a kind gift from Nevan Krogan ( ) (ORF8-Strep (Addgene #: 141390), Spike-Strep, eGFP-Strep (Addgene #: 141395)), Hyeran Choe ( ) (Spike-Flag (Addgene #: 156420), Spike-Flag FKO (Addgene #: 159364), Flag-Spike-Flag (Addgene #: 156418), and David Nemanzee ( ) (SARS-CoV Spike ΔC28 (Addgene #: 170447), Spike ΔC18 (Addgene #: 170442)).

Techniques: Infection, Virus, Transfection, Western Blot, Control, Stable Transfection, Expressing, Produced, Flow Cytometry, Incubation