c terminal c9 tagged sars cov 2 spike plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: High throughput screening identifies broad-spectrum Coronavirus entry inhibitors

doi: 10.1101/2023.12.04.569985

Figure Lengend Snippet: (A) A schematic representation of the glycoprotein complemented VSVΔG pseudoviruses producing process. Viral glycoproteins (yellow) are overexpressed on cell membranes using a plasmid encoding the chosen glycoproteins. These transfected cells are then infected with VSVΔG-G, resulting in viral-induced expression of a fluorescent reporter (FR) and VSV structural proteins that assemble on the cell surface, incorporating the desired glycoproteins into the VSVΔG pseudovirus. (B) A widefield image of cells infected with VSVΔG pseudoviruses expressing a fluorescent reporter (GFP; green). Infected cells become round after infection due to the virus-induced Cytopathic Effect. (C) A high magnification overlay image showing the infected GFP-positive cells (green) and total nuclei (blue), and the respective segmentation showing infected cells (yellow) and total nuclei (cyan). (D-G) Pseudoviral titers in infections/ml. An antibody against VSV-G (α-G) was utilized to neutralize residual VSVΔG-G infection from production, ensuring accurate titration of the heterologous pseudoviruses. (D) Titer of VSVΔG-S W showing enhanced infection of cells expressing the innate receptor, ACE2 and host protease, TMPRSS2. (E) Titer of VSVΔG-S W showing the effect of modifications to the cytosolic tail of S W . (F) Titer of VSVΔG-G, Wuhan (S W ), Alpha (S α ), Delta (S δ ), Omicron (S ο ), and MERS-CoV S (S M ) showing similar infection levels in HEK-293T cells expressing ACE2-TMPRSS2, with and without DPP4. (D-F) VSVΔG-G Pseudoviruses infected all cell lines at similar levels. (G) Pseudoviral infections/ml of VSVΔG RFP -G and VSVΔG GFP -S W separately or simultaneously results in equivalent infection rates. (Right) A high magnification overlay image of a well showing VSVΔG RFP -G and VSVΔG GFP -S W infected cells (White and green, respectively). The statistical significance of antibody activity was also determined. P: **≤0.01, ****≤0.0001 (two-tailed unpaired t-tests). N(experiments)=3, n(readings)=9. Error bars represent the SEM. Scale bar is 100 μM (B, C and G).

Article Snippet: pCAGGS-G, encoding the Vesicular Stomatitis Virus G glycoprotein from the Indiana serotype (VSV-G), was a kind gift from Benjamin Podbilewicz (Technion - Israel Institute of Technology) . pcDNA3.1-SARS2-Spike-C9, encoding the Spike glycoprotein of Wuhan SARS-CoV-2 fused to a C-terminal C9 tag (S W ) was a kind gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032 ; RRID:Addgene_145032) . pCG1-SARS2-Spike-HA, encoding the Wuhan SARS-CoV-2 Spike protein fused to a C-terminal HA tag was a kind gift from Gideon Schreiber, pCMV3-SARS2-Spike-Flag, encoding the Wuhan SARS-CoV-2 Spike protein fused to a C-terminal FLAG tag, pCMV3-SARS2-SpikeΔ19, encoding the Wuhan SARS-CoV-2 Spike protein with 19 amino acids removed at the cytoplasmic tail, and pCMV3-SARS2-SpikeΔ19-Flag, with an added C-terminal FLAG tag were a kind gift from and Yosef Shaul (Weizmann Institute of Science) . pcDNA3.1-SARS-CoV-2-S α , SARS-CoV-2-S δ , and SARS-CoV-2-S ο, encoding the Spike glycoprotein of Wuhan SARS-CoV-2 variants fused to a C-terminal C9 tag were generated by DNA synthesis (GeneScript). pcDNA3.1-MERS-Spike-C9, encoding the Spike glycoprotein of the MERS-CoV (S M ) fused to a C-terminal C9 tag was generated by sub-cloning the MERS Spike protein from a pLVX-EF1alpha-MERS-Spike plasmid (Weizmann Plasmid Bank) into a pcDNA3.1 expression plasmid using the GeneArt Gibson Assembly HiFi master mix (Thermo Fisher Scientific cat. no. A46627).

Techniques: Plasmid Preparation, Transfection, Infection, Expressing, Virus, Titration, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: High throughput screening identifies broad-spectrum Coronavirus entry inhibitors

doi: 10.1101/2023.12.04.569985

Figure Lengend Snippet: (A) Schematic showing primary screening of ∼200,000 compounds against Wuhan SARS-CoV-2 Spike (VSVΔG-S W ) yielded 733 putative Spike-specific and non-specific inhibitors. S W specific inhibitors were identified by a secondary screen against VSV-G (VSVΔG-G) yielding 65 putative inhibitors. The tertiary screen with MERS-CoV Spike (VSVΔG-S M ) and initial validation resulted in 5 compounds that were putatively broad-spectrum inhibitors. (B) Plots showing HTS parameters to determine the robustness of our screen: Z’ factor, signal-to-background, percentage of coefficient of variance for positive and negative for the primary screen. Red line denotes the cut off for a robust plate. Five plates that failed to meet the cut-off for Z prime were manually checked for data quality. (C) The chemical structures of the four novel compounds and Nafamostat that were commercially resourced. (D) Plot of the inhibition of the resourced compounds against the three viruses. All the compounds selectively inhibit SARS-CoV-2 and MERS-CoV Spikes without inhibiting VSV-G. Error bars represent the range. Values represent mean inhibitions ± standard deviations.

Article Snippet: pCAGGS-G, encoding the Vesicular Stomatitis Virus G glycoprotein from the Indiana serotype (VSV-G), was a kind gift from Benjamin Podbilewicz (Technion - Israel Institute of Technology) . pcDNA3.1-SARS2-Spike-C9, encoding the Spike glycoprotein of Wuhan SARS-CoV-2 fused to a C-terminal C9 tag (S W ) was a kind gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032 ; RRID:Addgene_145032) . pCG1-SARS2-Spike-HA, encoding the Wuhan SARS-CoV-2 Spike protein fused to a C-terminal HA tag was a kind gift from Gideon Schreiber, pCMV3-SARS2-Spike-Flag, encoding the Wuhan SARS-CoV-2 Spike protein fused to a C-terminal FLAG tag, pCMV3-SARS2-SpikeΔ19, encoding the Wuhan SARS-CoV-2 Spike protein with 19 amino acids removed at the cytoplasmic tail, and pCMV3-SARS2-SpikeΔ19-Flag, with an added C-terminal FLAG tag were a kind gift from and Yosef Shaul (Weizmann Institute of Science) . pcDNA3.1-SARS-CoV-2-S α , SARS-CoV-2-S δ , and SARS-CoV-2-S ο, encoding the Spike glycoprotein of Wuhan SARS-CoV-2 variants fused to a C-terminal C9 tag were generated by DNA synthesis (GeneScript). pcDNA3.1-MERS-Spike-C9, encoding the Spike glycoprotein of the MERS-CoV (S M ) fused to a C-terminal C9 tag was generated by sub-cloning the MERS Spike protein from a pLVX-EF1alpha-MERS-Spike plasmid (Weizmann Plasmid Bank) into a pcDNA3.1 expression plasmid using the GeneArt Gibson Assembly HiFi master mix (Thermo Fisher Scientific cat. no. A46627).

Techniques: Inhibition