Journal: bioRxiv

Article Title: Genome-scale CRISPR‒Cas9 screen identifies novel host factors as potential therapeutic targets for SARS-CoV-2 infection

doi: 10.1101/2023.03.06.531431

Figure Lengend Snippet: (a) Quantification of IFNβ mRNA at 24 hours post-infection. Poly:IC (HMW) was transfected as a positive control. (b) qPCR analysis of viral RNA. (c) Western blotting analysis of the viral nucleoprotein (N). The expression of viral protein was evaluated by the band intensity of N, which was normalized to that of GAPDH. Cell lysates (b, c) and culture supernatants (b) were harvested 3 days after infection. (d) VSV-based pseudotype assay. VSVΔG-GFP enveloped with VSV-G or SARS-CoV-2-S was used to infect knockout cells, and the number of GFP-positive cells was counted. (e) The subcellular localization of the spike protein in the infected cells. Cells were stained with anti-S antibody and phalloidin for visualization of cellular actin. Bar = 25 μm. (f) Electron microscopic analysis of virus particle formation in infected cells. Bar=5.0 μm. Data are presented as the mean ± the SD of three independent experiments. Statistical analysis was performed using Fisher’s LSD test. *, p < 0.05; **, p < 0.01. N.D., not detected.

Article Snippet: RNA was extracted from sgTRIM28, sgTRIM33, sgEHMT1, and sgEHMT2 cells using nucleospin RNA plus (TaKaRa), and RT‒qPCR was performed using Luna Universal qPCR Master Mix (New England Biolabs, New England Biolabs, Ipswich, MA, USA) and CFX96 (Bio-Rad) to quantify the mRNA levels of IFNb1 and CXCL10.

Techniques: Infection, Transfection, Positive Control, Western Blot, Expressing, Knock-Out, Staining

Journal: bioRxiv

Article Title: Genome-scale CRISPR‒Cas9 screen identifies novel host factors as potential therapeutic targets for SARS-CoV-2 infection

doi: 10.1101/2023.03.06.531431

Figure Lengend Snippet: (a) Quantification of mRNA for IFNβ and CXCL10 . Poly:IC (HMW) was transfected as a positive control. (b) qPCR analysis of viral RNA. (c) Western blotting analysis of viral N protein. (d) VSV-based pseudotype assay. (e) Inhibitory effect of the EHMT1/2-specific inhibitor UNC0642 upon SARS-CoV-2 infection. (f) qPCR analysis of viral RNA in UNC0642-treated cells. (g) Western blotting analysis of viral N protein in UNC0642-treated cells. The expression of viral protein was evaluated by the band intensity of N, which was normalized to that of GAPDH. Data are presented as the mean ± the SD of three independent experiments. Statistical analysis was performed using Fisher’s LSD test (b, c, f, g) and Dunnett’s multiple-comparison test (e). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: RNA was extracted from sgTRIM28, sgTRIM33, sgEHMT1, and sgEHMT2 cells using nucleospin RNA plus (TaKaRa), and RT‒qPCR was performed using Luna Universal qPCR Master Mix (New England Biolabs, New England Biolabs, Ipswich, MA, USA) and CFX96 (Bio-Rad) to quantify the mRNA levels of IFNb1 and CXCL10.

Techniques: Transfection, Positive Control, Western Blot, Infection, Expressing

Journal: bioRxiv

Article Title: SARS-CoV-2 ORF3c suppresses immune activation by inhibiting innate sensing

doi: 10.1101/2023.02.27.530232

Figure Lengend Snippet: (A) Genome organization of SARS-CoV-2 is illustrated on top; overlapping ORFs in the ORF3 locus are shown at the bottom. Vertical grey lines indicate internal ATG codons. Experimentally confirmed translation initiation sites ( Finkel et al ., 2021 ) are highlighted by grey triangles. (B) Kozak sequences of the ORF3c initiation codon and upstream ATG codons in ORF3a are shown. ATG context was determined using TIS predictor ( https://www.tispredictor.com/ ) ( Gleason et al ., 2022 ). (C) Western blot analysis of HEK293T cells transfected with two different concentrations of expression plasmids for the indicated ORF3 proteins and peptides. ORF3a to ORF3e were detected via a C-terminal HA tag. GAPDH served as loading control. (D) HEK293T cells were co-transfected with the indicated ORF3 expression plasmids, a reporter plasmid expressing firefly luciferase under the control of the IFNB1 promoter and a construct expressing Gaussia luciferase under the control of a minimal promoter. To induce immune signaling, half of the samples were additionally co-transfected with an expression plasmid for the CARD domain of RIG-I. One day post transfection, firefly luciferase activity was determined and normalized to Gaussia luciferase activity. Mean values of three independent experiments measured in triplicates (±SD) are shown. (E) HEK293T cells were transfected with an expression plasmid for SARS-CoV-2 ORF3c or an empty vector control. 24 hours post transfection, cells were infected with increasing amounts of Sendai virus (SeV) for an additional 8 hours. Cells were lysed to perform either RNA extraction and subsequent qPCR for IFN-β (left panel) or western blot analysis (right panel). Mean values of three independent experiments measured in duplicates (±SD) are shown. Multiple comparison within individual reporter assays (D) were determined by one-way ANOVA with Dunnett’s test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ % 0.001 and **** p ≤ 0.0001. Multiple comparison between groups (E) were determined by two-way ANOVA with Sidak’s multiple comparison test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ % 0.001 and **** p ≤ 0.0001.

Article Snippet: Genomic DNA was removed using the DNA-free kit (Thermo Fischer Scientific) and subsequent cDNA synthesis was performed using the PrimeScript RT reagent Kit (TAKARA), both according to the manufacturer’s instructions. qPCR was performed using the Luna Universal Probe qPCR Master Mix (NEB) together with primer probes for IFN-β, IL-6 and GAPDH (Thermo Fischer Scentific).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Luciferase, Construct, Activity Assay, Infection, RNA Extraction