Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: HuH7 cells were left untreated or were infected with HCoV-229E or MERS-CoV (MOI=1) for 3, 6, 12, or 24 h. Transcriptomic data (by RNA-seq, n=2) and proteomic data (by LC-MS/MS, n=2, three technical replicates per sample) were derived from samples obtained at the indicated time points p.i. and subsequently used to extract expression values for the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. (A) Scatter plots show mean normalized protein / mRNA expression values for each component, fitted linear regression lines, confidence intervals and correlation coefficients for mock-infected HuH7 cells and HuH7 cells infected for 24 h. (B) Correlation matrix of Pearson’s r across all conditions. All p values are given in supplementary Table S1. (C) The heatmap shows mean ratio values of differentially expressed mRNAs or proteins based on significant differences (fold change ≥ 2, p ≤ 0.01) calculated from the two biological replicates. See also and Table S1.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: Projection of normalized ratio values for transcriptomic (by RNA-seq) and proteomic (by LC-MS/MS) data derived in parallel from HuH7 cells infected for 24 h with HCoV-229E or MERS-CoV with a MOI=1 on the components of the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. The left side of the boxes show mRNA values, right sides show protein values.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Infection

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Schematic overview of parameters used to monitor virus- and thapsigargin-mediated ER stress. (B) Schematic presentation of HCoV-229E infection of cells and/or treatment with thapsigargin for different periods of time as applied in this study. (C) HuH7 cells were left untreated or infected with HCoV-229E (MOI=1) for 24 h and treated with thapsigargin (1 μM) according to (B). Supernatants and RNA-isolated from the cell pellets were used to determine viral titers (upper graphs) and expression of HCoV-229E S gene-containing RNA (lower graphs). (D) Representative fluorescence microscopy images showing subcellular HCoV-229E replication sites (at 24 h p.i.) identified by nsp8- or double-strand RNA-specific antibodies in the presence or absence of thapsigargin. Cells were infected with virus and treated with thapsigargin (1 μM) for 24 h. (E) Total cell extracts from HuH7 cells infected with HCoV-229E (MOI=1) and treated with thapsigargin (1 μM) as shown in (B) were analyzed for the expression/modification of the indicated host cell and viral proteins by immunoblotting. (F) Quantification of immunoblot data from multiple experiments. (G) HuH7 cells were left untreated, or were infected with HCoV-229E (MOI=1) in the presence / absence of increasing concentrations of thapsigargin or DMSO as solvent control. After 24 h, cell viability was assessed by MTS assay. (H) Data from (G) were used to compute half maximal cellular cytotoxicity (CC 50 ) concentrations of thapsigargin. (I) HuH7 cells were treated and infected according to (B). 30 min before harvesting, puromycin (3 μM) was added to the cells as indicated. Total cell extracts were analyzed by immunoblotting. The blot membrane was stained with CBB to assess the steady state proteomes and then hybridized with anti-puromycin antibodies to detect de novo synthesized nascent polypeptides. The upper graphs show representative images and the lower graph shows the quantification of multiple replicates. Puromycin signals of each lane were normalized to the corresponding CBB staining and were background corrected by subtracting signals of samples in which puromycin had been omitted. Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, Isolation, Expressing, Fluorescence, Microscopy, Modification, Western Blot, MTS Assay, Staining, Synthesized, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A-D) Human embryonic MRC-5 lung fibroblasts were infected with HCoV-229E according to the scheme shown in . Viral titers (A, upper graph), and expression of viral S gene-containing RNAs (A, lower graph), viral and host cell proteins (B, C) and cell viability (D) were analyzed and quantified as described in the legend of . (E-K) Similarly, HuH7 cells or Vero E6 African green monkey kidney epithelial cells were infected with MERS-CoV (MOI=0.5) or SARS-CoV-2 (MOI=0.5) for 12 h or 24 in the presence / absence of 0.4 μM or 1 μM thapsigargin. (E, F) show viral titers and (G, H) the corresponding expression of MERS-CoV / SARS-CoV-2 nucleocapsid (N) and host cell proteins, respectively. (I) Dose-dependent suppression of MERS-CoV-2 replication by thapsigargin (upper graph) and the estimated effective inhibitory concentration (EC 50 ) in HuH7 cells infected with an MOI of 0.5 (lower graph). (J) Dose-dependent suppression of SARS-CoV-2 replication by thapsigargin (left graph) and the calculated effective inhibitory concentration (EC 50 ) in Vero E6 cells infected with an MOI of 0.5 (right graph). (K) The CC 50 of thapsigargin in Vero E6 cells was calculated by MTS assays as described in the legend of . Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests. See for quantifications from replicates for MERS-CoV / SARS-CoV-2 immunoblot experiments.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, Expressing, Concentration Assay, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Venn diagram demonstrating the overlap of orthologues proteins expressed in HuH7 and Vero cells based on the NCBI gene IDs corresponding to majority protein IDs. (B) The overlap of virus- and thapsigargin-regulated proteins common to HuH7 and Vero E6 cells was calculated based on gene IDs. This analysis identifies 180 proteins with higher and 61 proteins with lower expression in thapsigargin-treated infected cells compared to virus infection alone (ratio > 0, p ≥ −log 10 1.3). (C) Heatmaps showing individual mean ratio values of log 2 -transfomed normalized protein intensities for the top 50 up- or downregulated proteins in virus-infected and thapsigargin-treated cells. Ratio values of infected or thapsigargin-treated conditions compared to untreated cells (-) cells are shown for comparison. Note that ratios are sorted and color coded according to the virus plus thapsigargin conditions with infection alone conditions set as denominator. Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2-regulated protein in an independent study ( Stukalov et al ., 2020 ). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone. Gene ID lists were analyzed using Metascape software ( Zhou et al ., 2019 ). (E) Experimental evidence, co-occurrence, co-expression and confidence scores from the STRING database ( Szklarczyk et al , 2019 ) were used to identify protein:protein interactions (PPI) amongst the 180 up- and 61 downregulated thapsigargin-sensitive proteins. As shown, based on experimental evidence and combined STRING score criteria, 59 and 26 coregulated proteins are engaged in defined PPI networks; the remaining proteins are not known to interact. (F) Validation of thapsigargin-induced HERPUD1 and CTH upregulation by immunoblotting of HuH7 or Vero E6 whole cell extracts from cells treated as described above. BiP and IRE1α levels are shown for comparison. (G) Quantification of thapsigargin-mediated re-expression of HERPUD1 and CTH in cells infected with HCoV-229E, MERS-CoV or SARS-CoV-2 from two independent immunoblot experiments. Error bars show s.d.. (H) Heatmap showing thapsigargin-reprogrammed proteins of KEGG 04141 (mean ratio ≥ 1.5 fold) along with p values. See also for projection of thapsigargin-mediated protein changes on the KEGG pathway map. (I) Venn diagram showing the intersection of thapsigargin- / virus-regulated proteins with all novel ERAD components (FDR of 1 %) identified by ( Leto et al ., 2019 ). The regulation of 31 overlapping components is shown as a heatmap displaying mean ratio values in thapsigargin-treated or infected cells. Red colors highlight UBA5 and ZNF622 as discussed in the text. (J) Summary of the main findings of our study. Abbreviations: M, MERS-CoV; S, SARS-CoV-2; T, thapsigargin.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Expressing, Infection, Software, Western Blot

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a Cartoon depicting the cell surface receptors involved in the viral entry for the coronaviruses SARS-CoV-2, 229E, and OC43, as well as potential viral replication steps that can be targeted by Cas13d. Cas13d is able to target both positive-sense genomic RNA as well as positive-sense subgenomic RNAs that are translated into proteins. The nested set of subgenomic RNAs is characteristic of the order Nidovirales , and means that the 3’-end of the genome that contains N is present in all subgenomic RNAs . b SARS-CoV-2 genome structure and the location of 23 crRNAs that are targeting the N gene, and which were evaluated for antiviral activity. c Inhibition of SARS-CoV-2 by Cas13d. Vero E6 cells expressing Cas13d and a single crRNA or a pool of crRNAs, using a non-targeting (NT) crRNA as a control, were challenged with SARS-CoV-2 USA-WA1/2020. At 24 hpi, the virus amount in the supernatant was measured by reverse transcription quantification PCR (RT-qPCR); n = 3, t = 3. d , e A summary of the kinetics of single ( d ) or pooled ( e ) crRNAs and their effect on virus replication; n = 3, t = 3. Data presented as means ± SEM. f Inhibition of SARS-CoV-2 variants by Cas13d. Vero E6 cells expressing Cas13d were infected with SARS-CoV-2 variants at a multiplicity of infection (MOI) of 0.01. At 48 hpi, the virus titer in the supernatant was determined by RT-qPCR; n = 3, t = 2. g The genome sequences of some SARS-CoV-2 variants were aligned with the targeting sequence of crRNA SN1 using Mafft v7.480. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for the virus-targeting crRNAs are relative to NT (refer to Supplementary Data ).

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified once by inoculating a T75 flask of fully confluent MRC-5 cells and collecting cell culture supernatant at 48 h post infection (hpi) followed by centrifugation at 1500× g for 10 min to remove cell debris.

Techniques: Activity Assay, Inhibition, Expressing, Control, Virus, Reverse Transcription, Quantitative RT-PCR, Infection, Sequencing

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a The genome structure of 229E. Includes the locations of the 20 crRNAs targeting the RdRp gene and the 20 crRNAs targeting the N gene. b – d Inhibition of 229E by Cas13d. At 48 hpi, the virus titer in the supernatant ( b ) was measured by RT-qPCR. Viral RNA abundance in cell lysates was quantified using two sets of primers targeting RdRp ( c ) and N ( d ) gene, respectively; n = 3, t = 3. e , f Cell viability after 229E challenge was measured using the Incucyte live imaging system. e Incucyte images showing brightfield and fluorescence overlay for selected time points. Dead cells are in green and Cas13d+ cells are in red; n = 3. f Quantification of the signal from dead cells over time; n = 3 wells per group, 9 images per well, per time point. Data presented as means ± SEM. g The genome structure of OC43 and the locations of the 10 N gene-targeting crRNAs. h Inhibition of OC43 by Cas13d. At 48 hpi, the viral genome copies in the supernatant were measured by RT-qPCR; n = 2, t = 3. i Phylogenetic tree of representative strains of SARS-CoV-2, 229E, OC43. The outer ring shows coverage by each of the top crRNAs targeting these three coronaviruses. j – l Co-infection of 229E and OC43 in MRC-5 cells expressing different combinations of crRNAs. j Schematic depicting the strategy to protect against both 229E and OC43 simultaneously. At 48 hpi, the 229E ( k ) or OC43 ( l ) virus titer in the supernatant was measured by RT-qPCR; n = 3, t = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for virus-targeting crRNAs (refer to Supplementary Data ) are relative to NT. APN aminopeptidase N, SA:9-O-ac 9-O-acetyl-modified sialic acid.

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified once by inoculating a T75 flask of fully confluent MRC-5 cells and collecting cell culture supernatant at 48 h post infection (hpi) followed by centrifugation at 1500× g for 10 min to remove cell debris.

Techniques: Inhibition, Virus, Quantitative RT-PCR, Imaging, Fluorescence, Infection, Expressing, Modification

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a MRC-5 cells, transduced with the indicated Cas13d (with or without a subcellular location signal tag) and the NT or N1 crRNA, were challenged with 229E. Viral titer was determined by RT-qPCR; n = 3, t = 2. b Fluorescent microscopy of MRC-5 cells expressing mCherry-fused NES or NLS-Cas13d along with the NT or N20 crRNA, infected by 229E and fixed at 24 hpi. The 229E viral RNA was stained with RNA FISH probes labeled with AF647. c FISH signal (229E viral RNA abundance) mean intensity of each cell. All cells quantified were mCherry (Cas13d) and blue fluorescent protein (BFP; indicates crRNA + cells) double positive. d , e The FISH signal and cytosol mCherry (Cas13d) signal measured from cells expressing NLS-Cas13d and N20 ( d ) and cells expressing NES-Cas13d and N20 ( e ). f MRC-5 cells, expressing NES or NLS-Cas13d, were delivered with indicated crRNA using lentivirus or lipid nanoparticle (LNP), and challenged with 229E. At 24 hpi, viral RNA in the supernatant was measured by RT-qPCR; n = 3 (for lentiviral delivered crRNA, t = 2; for LNP delivered crRNA, t = 3). g Cas13d antiviral activity against 229E infection at different MOIs; n = 3. n is the number of independent biological experiments. t is the number of technician repeats per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified once by inoculating a T75 flask of fully confluent MRC-5 cells and collecting cell culture supernatant at 48 h post infection (hpi) followed by centrifugation at 1500× g for 10 min to remove cell debris.

Techniques: Transduction, Quantitative RT-PCR, Microscopy, Expressing, Infection, Staining, Labeling, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a The life cycle of coronavirus and known/hypothesized mechanism of action of antivirals targeting the different pathways involved in viral replication. b Combinations of Cas13d with antiviral small-molecule compounds and their effect on inhibition of SARS-CoV-2. Vero E6 cells expressing Cas13d and NT or SN1 crRNA were pre- and post-treated with indicated drug and challenged with USA-WA1/2020 SARS-CoV-2 at an MOI of 0.01. At 48 hpi, the viral genome copies in the supernatant were determined by RT-qPCR; n = 3, t = 3. c Combinations of Cas13d with small-molecule antivirals on inhibition of 229E virus. MRC-5 cells expressing Cas13d and NT or N1 crRNA were pre- and post-treated with the indicated antiviral drugs at the indicated dose and infected with 229E at an MOI of 0.01. The virus titer was measured at 48 hpi by RT-qPCR; n = 3, t = 3. d The combination of Cas13d with several drugs at a dose of EC15 on inhibition of 229E virus; n = 3, t = 3. e Combination of 229E- and host gene-targeting crRNAs on the inhibition of 229E; n = 3, t = 2. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data .

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified once by inoculating a T75 flask of fully confluent MRC-5 cells and collecting cell culture supernatant at 48 h post infection (hpi) followed by centrifugation at 1500× g for 10 min to remove cell debris.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Virus, Infection

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a Schematic of the antiviral treatment of SARS-CoV-2 infection in Vero E6/NES-Cas13d cells. b Treatment test of Cas13d targeting SARS-CoV-2 in Vero E6/NES-Cas13d cells. At 6 hpi, the cells were transfected with crRNA (Synthego), NT or SN1, using LNP. At 48 hpi, the virus titer in the media was quantified using both RT-qPCR and plaque assays; n = 4, t = 3. d The antiviral drug EIDD-1931 was added to the media in combination with the Cas13d; n = 3, t = 3. c , e – g Treatment test of Cas13d targeting 229E in MRC-5 cells. At 0, 1, 3, and 6 hpi, the cells were transfected with NES-Cas13d mRNA (Trilink) and crRNA (Synthego) using lipofectamine MessagerMAX (Invitrogen). The indicated small-molecule antiviral drug was added to the media 20 min after the transfection. At 48 hpi, virus titer in the supernatant was measured by RT-qPCR. The viral genomic copies of Cas13d treatment with no drug ( c ) or combined with a small-molecule drug ( e ) are plotted separately; n = 3, t = 3. At 72 hpi, the cells were stained with crystal violet ( f ) and the percent vial cells were quantified using Fiji ImageJ ( g ); n = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified once by inoculating a T75 flask of fully confluent MRC-5 cells and collecting cell culture supernatant at 48 h post infection (hpi) followed by centrifugation at 1500× g for 10 min to remove cell debris.

Techniques: Infection, Transfection, Virus, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a Scheme used for antiviral treatment of air–liquid-interface (ALI) cultures of human primary bronchial epithelial cells (hPBECs). hPBECs were transduced with NES-Cas13d and differentiated to ALI cultures. The ALI cultures were infected with 229E at an MOI of 0.05, with SARS-CoV-2 WA1 at an MOI of 0.6, or with Omicron at an MOI of 0.1. At 6 hpi, the culture was transfected with crRNA using LNP. At 48, 72 hpi, the apical surfaces of ALI cultures were washed with PBS and the wash solution was collected for measuring virus titer. b The titer of 229E virus released on the apical surface of ALI cultures was determined by RT-qPCR; n = 4, t = 3. c The titer of SARS-CoV-2 virus, including WA1 and Omicron strains, was determined at 48 hpi; n = 3, t = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified once by inoculating a T75 flask of fully confluent MRC-5 cells and collecting cell culture supernatant at 48 h post infection (hpi) followed by centrifugation at 1500× g for 10 min to remove cell debris.

Techniques: Transduction, Infection, Transfection, Virus, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific Reports

Article Title: Cross-reactive antibodies elicited to conserved epitopes on SARS-CoV-2 spike protein after infection and vaccination

doi: 10.1038/s41598-022-10230-y

Figure Lengend Snippet: Vaccination targeting SARS-CoV-2 did not significantly affect antibody responses against most seasonal coronaviruses. ( A ) Bar and line graph of antibody responses obtained by ELISA at different plasma dilutions to Human CoV OC43 spike protein, before the Pfizer-BioNTech BNT162b2 vaccine was administered (Week 0) and after both doses (Week 7), using plasma from patients with no history of SARS-CoV-2 infection, n = 24 individuals, p ≤ 0.0001. ( B ) Bar and line graph of antibody responses obtained by ELISA at different plasma dilutions to Human CoV HKU1 spike protein, before the Pfizer-BioNTech BNT162b2 vaccine was administered (Week 0) and after both doses (Week 7), using plasma from patients with no history of SARS-CoV-2 infection, n = 24 individuals, p ≥ 0.99. ( C ) Bar and line graph of antibody responses obtained by ELISA at different plasma dilutions to Human CoV 229E spike protein, before the Pfizer-BioNTech BNT162b2 vaccine was administered (Week 0) and after both doses (Week 7), using plasma from patients with no history of SARS-CoV-2 infection, n = 24 individuals, p = 0.75. ( D ) Bar and line graph of antibody responses obtained by ELISA at different plasma dilutions to Human CoV NL63 spike protein, before the Pfizer-BioNTech BNT162b2 vaccine was administered (Week 0) and after both doses (Week 7), using plasma from patients with no history of SARS-CoV-2 infection, n = 24 individuals, p ≤ 0.0001. Endpoint titers are designated by the most dilute plasma concentration detected above the minimum threshold of the background OD450 multiplied by 3. Dots are representative of individual samples. Bars are representative of the mean of all samples. Statistical analysis was performed using Wilcoxon matched-pairs signed rank test with two-tailed P values reported.

Article Snippet: ELISAs were performed using the following antigens: SARS-CoV-1 (Cat# 10683-CV, Lot# DOXT0120121, R&D Systems, Minneapolis, MN, USA), SARS-CoV-2 (Cat# 10549-CV, Lot# DODR0221011, R&D Systems), Bat CoV RaTG13 (Cat# 10660-CV, Lot# DOWW0120121, R&D Systems), MERS-CoV spike protein (Cat# 40069-V08B, Lot# LC14AP2305, Sino Biological, Wayne, PA, USA), Human CoV NL63 spike protein (Cat# 40600-V08H, Lot#LC14NO2607, Sino Biological), Human CoV 229E spike protein (Cat# 40605-V08H, Lot# LC14AP2302, Sino Biological), Human CoV HKU1 spike protein (Cat# 40021-V08H, Lot# LC14AP2707, Sino Biological), Human CoV OC43 spike protein (Cat# 40607-V08H, Lot#LC14DE1609, Sino Biological) were all diluted to 1 ug/mL in 0.1 M sodium bicarbonate and incubated on high-binding plates (3369, Corning Inc, Corning, NY, USA) overnight at 4 degrees.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Concentration Assay, Two Tailed Test

Journal: Journal of Virological Methods

Article Title: Host-encoded reporters for the detection and purification of multiple enveloped viruses

doi: 10.1016/j.jviromet.2010.04.002

Figure Lengend Snippet: Alkaline phosphatase can be incorporated into multiple enveloped viruses. (A) Vero reporter cell lines were infected with Respiratory Syncytia Virus (RSV) and assayed for alkaline phosphatase (AP) activity in supernatants after 7 days. (B) The presence of RSV was confirmed with the respiratory detection kit (Chemicon) by orange-red fluorescence specific for RSV (lower panel). (C) Vero reporter cell lines were infected with Herpes Simplex Virus 1 (HSV1) and cultured for up to 3 days. Supernatants were collected, centrifuged and analyzed for alkaline phosphatase activity. (D) Vero cells were infected with HSV1 as in (C). After 24 h, cell rounding was observed as a hallmark of efficient infection with HSV1 by light microscopy in infected cells (lower panel) compared to uninfected cells (upper panel). The presence of HSV1 was confirmed by immunoblotting with antibodies raised against HSV1 (lower panel). (E) MRC5 cells transduced with GFP or FPLAP were infected with human coronavirus 229E in 2% serum at 35 °C. After 3 days, supernatants were analyzed for alkaline phosphatase activity. (F) Cytophathic effects were visible after 3 days in cells infected with HCoV 229E (lower panel) compared to the confluent layer of GFP-positive cells in uninfected MRC5 cells (upper panel). The cells were lysed and subjected to immunoblotting with anti-HCoV antibody (Chemicon, as recommended by ATCC) that is highly specific for the 229E sub-type of HCoV, but not for OC43. A single band was detected only in cells that were infected with HCoV 229E. Results from three independent infections + standard deviation (SD) are shown. RLU, relative light units. * p < 0.05; *** p < 0.001; Student's t -test, paired, two-tailed.

Article Snippet: Human respiratory syncytial virus (RSV; strain B WV/14617/’85; ATCC, #VR-1400), Aguacate virus (ATCC, #VR900), and human herpesvirus HSV1, strain F (ATCC, #VR-733) were propagated in Vero cells maintained in 2% calf serum (supplemented with iron) at 35 °C.

Techniques: Infection, Virus, Activity Assay, Fluorescence, Cell Culture, Light Microscopy, Western Blot, Transduction, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a Scheme used for antiviral treatment of air–liquid-interface (ALI) cultures of human primary bronchial epithelial cells (hPBECs). hPBECs were transduced with NES-Cas13d and differentiated to ALI cultures. The ALI cultures were infected with 229E at an MOI of 0.05, with SARS-CoV-2 WA1 at an MOI of 0.6, or with Omicron at an MOI of 0.1. At 6 hpi, the culture was transfected with crRNA using LNP. At 48, 72 hpi, the apical surfaces of ALI cultures were washed with PBS and the wash solution was collected for measuring virus titer. b The titer of 229E virus released on the apical surface of ALI cultures was determined by RT-qPCR; n = 4, t = 3. c The titer of SARS-CoV-2 virus, including WA1 and Omicron strains, was determined at 48 hpi; n = 3, t = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

Article Snippet: Human primary bronchial epithelial cells (hPBEC) were purchased from ATCC (PCS300010) and maintained in PneumaCult-Ex Plus Medium (StemCell, Canada) supplemented with hydrocortisone (0.5 μg/ml).

Techniques: Transduction, Infection, Transfection, Virus, Quantitative RT-PCR, Two Tailed Test

Journal: Membranes

Article Title: Virucidal and Bactericidal Filtration Media from Electrospun Polylactic Acid Nanofibres Capable of Protecting against COVID-19

doi: 10.3390/membranes12060571

Figure Lengend Snippet: ( left ) Antibacterial activity of PLA NF filter media containing manuka triketone (5 wt.%) against S. aureus (ATCC 6538P), E. coli (ATCC 8739) and K. pneumoniae (ATCC 4352) tested according to ISO 20743:2013 . ( right ) Antiviral activity of PLA NF filter media containing manuka triketone (5 wt.%) against influenza A, human coronavirus 229E and SARS-CoV-2 tested according to ISO 18184:2019 . The arrows point at the inhibition of microbial growth.

Article Snippet: The electrospun PLA filter media were challenged against the test standard ISO 18184:2019 [ ] using three different viruses: influenza A [H1N1] (ATCC VR-1469), human coronavirus 229E (ATCC VR-740) and SARS-CoV-2 (Delta variant B.1.617.2; NCBI MZ574052) ( , right and ).

Techniques: Activity Assay, Inhibition

Journal: Membranes

Article Title: Virucidal and Bactericidal Filtration Media from Electrospun Polylactic Acid Nanofibres Capable of Protecting against COVID-19

doi: 10.3390/membranes12060571

Figure Lengend Snippet: Antibacterial reduction rate of PLA NF filter media containing manuka triketone (5 wt.%) against S. aureus (ATCC 6538P), E. coli (ATCC 8739). and K. pneumoniae (ATCC 4352) tested according to ISO 20743:2013 [ 43 ]. Reusability following 5 and 10 laundering cycle according to ISO 6330:2013 [ 49 ] and ISO 20743:2013, was confirmed for the PLA NF filter media when tested against S. aureus (ATCC 6538P) and K. pneumoniae (ATCC 4352).

Article Snippet: The electrospun PLA filter media were challenged against the test standard ISO 18184:2019 [ ] using three different viruses: influenza A [H1N1] (ATCC VR-1469), human coronavirus 229E (ATCC VR-740) and SARS-CoV-2 (Delta variant B.1.617.2; NCBI MZ574052) ( , right and ).

Techniques: Control

Journal: Membranes

Article Title: Virucidal and Bactericidal Filtration Media from Electrospun Polylactic Acid Nanofibres Capable of Protecting against COVID-19

doi: 10.3390/membranes12060571

Figure Lengend Snippet: Antiviral reduction rate of PLA NF filter media containing manuka triketone (5 wt.%) against influenza A, human coronavirus 229E and SARS-CoV-2 tested according to ISO 18184:2019 [ 44 ].

Article Snippet: The electrospun PLA filter media were challenged against the test standard ISO 18184:2019 [ ] using three different viruses: influenza A [H1N1] (ATCC VR-1469), human coronavirus 229E (ATCC VR-740) and SARS-CoV-2 (Delta variant B.1.617.2; NCBI MZ574052) ( , right and ).

Techniques: Variant Assay, Plaque Assay, Control, Activity Assay

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , Alignment of functionally important ISG15 residues. The sequence logos show the protein conservation of mammalian orders. b , Representative western blot of HEK293-subclone-ΔISG15 cells overexpressing Myc-tagged wild-type or mutated ISG15 . Lysates were treated with 1,6-bis(maleimido)hexane (BMH) to cross-link disulfide bonds. c , Quantification of ISG15 western blots. Two-tailed t -tests test for differences in dimer formation; n = 4. d – g , Viral output, measured by TCID 50 ( f and g ) or a TCID 50 surrogate (Crystal Violet absorbance at 450 nm; d and e ) in a HEK293-subclone ( d ), Vero-E6 ( e ), stable ANPEP-expressing HEK293 cells ( f ) and stable ACE2-expressing A549 cells ( g ) that express ISG15 constructs. d , VSV–GFP infection (multiplicity of infection (MOI) of 0.1 for 24 h); n = 3. e , H1N1 PR8 IAV infection (MOI of 0.1 for 48 h); n = 4. f , HCoV-229E infection (MOI of 0.1 for 72 h); n = 3. g , SARS-CoV-2 infection (MOI of 0.1 for 48 h); n ≥ 3 for ISG15, n = 7 for SARS-CoV-2 control. h , Quantification of western blots (Extended Data Fig. ) of ISG15-transfected HEK293-subclone-ΔISG15 cells, co-transfected with SARS-CoV-2 PLpro (NSPC3C/L); n = 4. i , Quantification of ISG15 conjugated in cells after PLpro cleavage (matched to h ). Decrease in conjugation with PLpro indicates de-ISGylation; n = 4. j , ISG15 released into the supernatant after PLpro cleavage (matched to h ). In supernatants, ISG15 is not detected before infection; n = 4. k , Quantification of western blots for ISG15 cleavage and release into the supernatant (example in j ), on a logarithmic scale; n = 4. Data are presented as mean (solid oval) and standard error of the mean (s.e.m.) (bars), showing individual data points of biological replicates as grey circles ( c – g , h , i and k ). All quantifications are from independent experiments. Significant differences to vector control (blue asterisks) or wild-type ISG15 (black asterisks) were determined by two-tailed t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). Uncropped images, P values and all data are in and Supplementary Tables , , , and – . WT, wild type; C78A, Cys78 to Ala mutation; ΔC78, Cys78 deletion; S77C, Ser77 to Cys mutation. All animal silhouettes apart from the bat were obtained from PhyloPic ( phylopic.org ). Mus musculus , by Daniel Jaron under a CC0 1.0 Universal Public Domain licence; Gorilla gorilla gorilla by T. Michael Keesey (after Colin M. L. Burnett) under a CC0 1.0 Universal Public Domain licence; Leporidae , by Sarah Werning under a CC BY 3.0 licence; Erinaceus europaeus by Roberto Díaz Sibaja under a CC BY 3.0 licence; Equus ferus przewalskii , by Mercedes Yrayzoz (vectorized by T. Michael Keesey) under a CC BY 3.0 licence; Camelus dromedarius , by Steven Traver under a CC0 1.0 Universal Public Domain licence; Panthera pardus , by Margot Michaud under a CC0 1.0 Universal Public Domain licence; Manis culionensis , by Steven Traver under a CC0 1.0 Universal Public Domain licence. The bat silhouette was vectorized by A.E.M., the Afrotheria (Thai Elephant) silhouette was obtained from OpenClipArt ( https://openclipart.org/ ) under a CC0 1.0 Universal Public Domain licence.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Sequencing, Western Blot, Two Tailed Test, Expressing, Construct, Infection, Control, Transfection, Conjugation Assay, Plasmid Preparation, Mutagenesis

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Infection, Western Blot, Transfection, Control, Two Tailed Test, Conjugation Assay, Plasmid Preparation, Expressing, Construct

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a – c , Viral output, expressed relative to the empty vector (IRES–mCherry) control, in A549 ( a ), stable ANPEP-expressing HEK293 ( b ) and stable ACE2-expressing A549 ( c ) cells that express ISG15 constructs. a , H1N1 PR8 IAV infection (as per Fig. ). A549 cell supernatants titrated on Vero-E6 cells by plaque assays; n = 4. b , HCoV-229E infection (as per Fig. ). Viral output was measured by TCID 50 in Huh7 cells; n = 3. c , SARS-CoV-2 infection (as per Fig. ). Viral output from A549 cells was measured by TCID 50 in Vero76 cells; n ≥ 4 for ISG15, n = 7 for SARS-CoV-2 control. d , As c except using wild-type (WT) and LRGG>LRAA ISG15 mutants that remove the ISG15 conjugation motif; n ≥ 5 for LRGG mutations, n = 7 for SARS-CoV-2 control. Graphs represent mean and s.e.m. with individual data points in grey; n refers to independent biological replicates. Significant differences to vector control (black asterisks) or human ISG15 (orange asterisks) were determined by two-tailed t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). All data are given in Supplementary Tables – .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Plasmid Preparation, Control, Expressing, Construct, Infection, Conjugation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Virus-derived Synthetic Riboswitches for Real-time Coronaviruses Sensing in Live Cells

doi: 10.1101/2024.02.27.582433

Figure Lengend Snippet: (a) The genomic and subgenomic vRibo designs encoding both GFP and mRuby3. (b-d) The expression of GFP and mRuby3 in the 293T/hAPN cells with or without 229E infection at 48 hpi. 4 independent biological replicates were performed with 8 images per biological replicate. The bar represents the average of the group, while each circle represents an individual technical repeat. P values were calculated by two-tailed Student’s t-tests. (e) The design of vRibos of SARS-CoV-2, 229E, and OC43 with a packaging sequence. (f) The GFP signal observed in 293T/hAPN cells upon re-infection with virions collected from the media of 293T/hAPN cells without transfection (negative control, NC) or transfected with vRibos (S, E, O) and either mock-infected or infected with 229E or OC43. 4 independent biological replicates were performed with 4 images per biological replicate. The bar represents the average of the group, while each circle represents an individual image. P values were calculated by one-tailed Student’s t-tests. (g) The fold activation of the GFP signal in the infected vs mock cells expressing vRibo (−) of 229E, OC43, and SARS-CoV-2, of which the source data is plot in . n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified and tittered using MRC-5 cells as described previously .

Techniques: Expressing, Infection, Two Tailed Test, Sequencing, Transfection, Negative Control, One-tailed Test, Activation Assay

Journal: bioRxiv

Article Title: Virus-derived Synthetic Riboswitches for Real-time Coronaviruses Sensing in Live Cells

doi: 10.1101/2024.02.27.582433

Figure Lengend Snippet: (a) The vRibo designs of human coronavirus OC43 and (b) test of the activation of the reporter expression with OC43 infection. A total of 32 images, divided into 4 separate biological replicates, were collected and the integrated GFP fluorescence intensity was calculated. (c) The design of the RNA sensors of SARS-CoV-2 and (d) test of their effectiveness with SARS-CoV-2 infection. The luciferase activity was measured with 3 independent biological replicates. (e-g) The reporter activation activity of vRibos of 229E, OC43, and SARS-CoV-2 by 229E (e), OC43 (f) , and SARS-CoV-2 infection. 4 independent biological replicates were performed with 2 images collected per biological replicate (e-f). The luciferase activity was measured with 3 independent biological replicates (g). Data are presented as mean ± s.e.m. P values were calculated by two-tailed Student’s t-tests. n.s., not significant; **P < 0.01; ***P < 0.001.

Article Snippet: Human coronavirus 229E (Cat# VR-740, ATCC) and OC43 (Cat# VR-1558, ATCC) were amplified and tittered using MRC-5 cells as described previously .

Techniques: Activation Assay, Expressing, Infection, Fluorescence, Luciferase, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: ZBTB7A promotes virus-host homeostasis during human coronavirus 229E infection.

doi: 10.1016/j.celrep.2022.111540

Figure Lengend Snippet: Figure 1. CRISPR activation screen identifies host factors that allow avoidance of HCoV-229E-induced cell death (A) Overview of CRISPR activation screen. (B) Distribution of sgRNA reads before (unselected) and after (post-selected) infection; the most abundant 500 sgRNAs after selection are shown. (C) The top seven enriched genes based on the inverse of the p value. (D) Quantification of mRNA by qRT-PCR from the indicated overexpression cell lines. n = 4 biological replicates. ND, not detected. (E) Cell viability of transduced Huh7 cells after viral infection. Transduced cells were infected with HCoV-229E at 0.5 MOI. MTT assay was conducted 3 DPI. n = 4 biological replicates. (F) Crystal violet staining of transduced Huh7 cells after infection. Control Huh7 cells expressing mCherry, ZBTB7A-Huh7 cells, ZBTB7B-Huh7 cells, and GEMIN7-Huh7 cells were infected with HCoV-229E at 0.5 MOI. The cells were stained at 5 DPI. n = 4 biological replicates. All panels except (A)–(C) are representative of two independent experiments. For (D) and (E), p values were calculated by unpaired two-tailed Student’s t tests. *p < 0.05; **p < 0.001; ns, not significant. Data shown as mean ± SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-ZBTB7A Santa Cruz Cat# sc-33683; RRID:AB_668999 anti-ZBTB7A R&D Cat# mab3496; RRID: AB_10584995 anti-alpha tubulin Abcam Cat# ab179484; RRID:AB_2890906 anti-alpha actin Santa Cruz Cat# sc-47778; RRID:AB_626632 anti-HCoV-229E N Eurofins Technologies Cat# M.30.HCo.I1E8 anti-Flag Sigma-Aldrich Cat# F1804; RRID:AB_262044 anti-GFP Cell Signaling Technology Cat# 2955; RRID:AB_1196614 anti-rabbit-HRP Thermo Fisher Scientific Cat# A16104; RRID:AB_2534776 anti-armenian hamster-HRP Novus Biologicals Cat# NB100-2066; RRID:AB_10000771 Alexa Fluor 594 Goat Anti-Mouse Thermo Fisher Scientific Cat# A11005; RRID:AB_2534073 Bacterial and virus strains Human coronavirus 229E ATCC VR-740 strain Chemicals, peptides, and recombinant proteins Puromycin Sigma-Aldrich Cat# P5412 Blasticidin S Sigma-Aldrich Cat# SBR00022 Poly-L-lysine solution Sigma-Aldrich Cat# P8920 Hoechst 33342 Invitrogen Cat# H3570 Universal SYBR Green Supermix Bio-Rad Cat# 1725274 MTT solution Abcam Cat# ab211091 Caspase 3/7 Green Detection Reagent Thermo Fisher Scientific Cat# C10423 CellROX Green Reagent Thermo Fisher Scientific Cat# C10444 Z-VAD-FMK R&D Cat# FMK001 Ac-DEVD-CHO Selleck Cat# S7901 Tiron Abcam Cat# ab146234 Trolox Sigma-Aldrich Cat# 53188-07-1 Hydrogen peroxide solution Sigma-Aldrich Cat# 7722-84-1 Critical commercial assays QIAamp DNA Blood Maxi Kit QIAGEN Cat# 51192 NEB Monarch total RNA miniprep kit NEB Cat# T2010 SuperScriptTM III One-Step RT-PCR Thermo Fisher Scientific Cat# 12574026 HiFi DNA assembly NEB Cat# M5520AA One-Step SuperscriptTM qRT-PCR Thermo Fisher Scientific Cat# 11781200 EdU Assay Kit Abcam Cat# ab219801 LIVE/DEAD Fixable Green Dead Cell Stain Kit Thermo Fisher Scientific Cat# L34969 LIVE/DEAD Cell Imaging Kit Thermo Fisher Scientific Cat# R37601 Poly(A) messenger RNA Magnetic Isolation Module NEB Cat# E74905 Ultra II RNA Library Prep Kit NEB Cat# E7770S PierceTM Immunoprecipitation, Magnetic ChIP Kit Thermo Fisher Scientific Cat# 26157 Deposited data CRISPR sequencing data This paper GEO: GSE197882 RNA sequencing data This paper GEO: GSE197644 Experimental models: Cell lines Huh7 cells Collaborating Laboratory, Duke University (US) N/A HEK-293T cells ATCC CRL-3216 (Continued on next page) e1 Cell Reports 41, 111540, October 25, 2022

Techniques: CRISPR, Activation Assay, Infection, Selection, Quantitative RT-PCR, Over Expression, MTT Assay, Staining, Control, Expressing, Two Tailed Test