wild type human coronavirus hcov oc43  (ATCC)

Journal: bioRxiv

Article Title: Proximity proteomics reveals a role for IFI16 during human coronavirus infection

doi: 10.64898/2026.04.22.720112

Figure Lengend Snippet: IFI16 promotes coronavirus infection. ( a) CRISPR-Cas9 technology was used to knock out IFI16 in A549 cells and single cell clones were screened for loss of IFI16 expression using qRT-PCR. Data is representative of two independent experiments. ( b) Immunoblot showing the loss of IFI16 expression in IFI16-KO cell line. ( c) Immunofluorescence assay showing reduced number of HCoV-OC43 N-positive cells upon loss of IFI16. Cells were infected at an MOI of 0.1 and incubated for the indicated length of time before fixation and staining for HCoV-OC43 N protein. Scale bar is 10 um. ( d ) Representative flow cytometry analysis of wildtype (WT) A549 cells and IFI16-KO cells infected (in quadruplicate) with HCoV-OC43 at an MOI of 0.01 and 0.1 for 12, 24 and 48 h. Data is representative of 3 independent experiments. (e) and (f) Quantification of flow cytometry data in . ( g) Representative flow cytometry analysis of CRISPRi cell lines infected (in quadruplicate) with HCoV-OC43 at an MOI of 0.01 for 12, 24 and 48 h. data in figure. (h) Quantification of flow cytometry data in . Data is representative of two independent experiments. Statistical significance was assessed by ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Cells were washed twice with a permeabilization/wash buffer (0.1% saponin and 0.5% BSA in 1X PBS) and stained with a 1:10000 dilution of anti-HCoV-OC43-N rabbit polyclonal antibody (catalogue no. 40643-T62; Sino Biological) for 30 min at 4°C.

Techniques: Infection, CRISPR, Knock-Out, Single Cell, Clone Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Incubation, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): A549 clone 2E10 was treated with IFN⍺/IFNγ for 16h then infected as indicated. Infection was assessed 72 hpi by flow cytometry. (B): RNA-seq analysis heatmap depicting normalized expression of selected ISGs across four A549 SCCs (1B7, 1C10, 2E10, 3G7) after 16h mock/IFNα/IFNγ treatment. TPM; transcripts per million. (C): Venn diagram of overlap between differentially expressed genes (>4-fold change and padj < 0.05) following IFNα/IFNγ treatment. (D): Schematic of screen setup. (E): Screen results, where the x axis corresponds to each unique gene in the library, and the y axis denotes the relative fold change. Differentially represented genes in infected versus uninfected populations (p<0.01) are labeled in magenta, non-targeting controls are labeled in blue. (F): Flow cytometry of knockout cells treated with IFNα or IFNγ for 16h and infected with HCoV-OC43 for 72h (normalized to EV control).

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Infection, Flow Cytometry, RNA Sequencing, Expressing, Labeling, Knock-Out, Control

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): RNF213 KO (n=20) or EV (n=7) A549 SCCs were treated as indicated for 16h, infected with HCoV-OC43 at MOI 0.1 for 72h and percent infected cells determined by flow cytometry. Each dot represents an independent SCC (representative experiment, n=3; statistics used each clone as an data point, +/- SD, p values shown: student’s t-test). (B) : Growth curve of HCoV-OC43 in A549 EV or RNF213 KO cells pretreated, or not, for 16h with IFNγ and infected at MOI 0.1 (n=3). (C): Cells were pretreated with IFNγ for 16h and infected with HCoV-OC43 at MOI 5 for 24h. Labeled extracellular virions were run on an SDS-PAGE gel (representative image, n=3). (D): A549 cells were transfected with siRNAs against RNF213 or a non-targeting control (NT), pretreated with IFNγ for 16h, and plaque assay performed for rHCoV-OC43/eGFP (representative image, n=3, scale 1 mm). (E): Quantification of plaque size (representative experiment shown +/- SD, p values shown: 2-way ANOVA). (F): Quantification of plaque number (n=3, +/- SD, p values shown: 2-way ANOVA). (G): siRNA transfected A549 cells were pretreated or not with IFNγ and infected with HCoV-OC43. Percentage infected cells at 72 hpi was determined by flow cytometry (n=3 +/-SD), (H): Same as (G) , in Vero cells (I): Schematic of human RNF213 and locations of CRISPR-mediated editing. (J): Flow cytometry of A549 SCCs containing the indicated RNF213 mutations, pretreated with IFNγ (16h) and infected with HCoV-OC43 at MOI 0.1 for 72h. Each dot represents an independent SCC (representative experiment, n=3; statistics based on each clone as an independent experiment, +/- SD, p values shown: 2-way ANOVA). EV: empty vector. KO: knockout. MOI: multiplicity of infection. PFU: plaque forming units. Tri: Trimer. Mono: Monomer. SCC: single-cell clone.

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Infection, Flow Cytometry, Labeling, SDS Page, Transfection, Control, Plaque Assay, CRISPR, Plasmid Preparation, Knock-Out, Single Cell

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): Quantification of fluorescent signal for each of the indicated bands from (n=3, +/- SD, p-values displayed: student’s t-test). (B): Calculation of the ratio of each other protein to M (n=3, +/- SD, p-values displayed: student’s t-test). (C): Flow cytometry analysis of A549 RNF213 knock-out cells containing knock-in mutations in the guide RNA target sequence. Cells were pretreated for 16h with IFNγ and infected with HCoV-OC43 at MOI 0.1 for 72 hours (n=3, +/- SD, p-values shown: 2-way ANOVA). (D): Western blot of EV or RNF213 KO A549 cells pretreated with IFN for 24 hours (representative images shown, n=3). (E): Western blot of the indicated cells transfected with siRNAs against RNF213 or a non-targeting control, then pretreated with IFNγ for an additional 24 hours (representative images shown, n=3). (F-G): Flow cytometry of the indicated cells transfected with siRNAs, pretreated with IFNγ for 16 hours, and infected with HCoV-OC43 (F) or the indicated virus (G) at the indicated MOI for 72h, except VSV which was infected for 24h (n=3. +/- SD). (H): Western blot of A549 cells with the indicated point mutations pretreated with IFNγ for 24 hours (representative images shown, n=3).

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Flow Cytometry, Knock-Out, Knock-In, Sequencing, Infection, Western Blot, Transfection, Control, Virus

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): Flow cytometry analysis of A549 cells transfected with siRNAs, treated with IFNγ for 16 hours, and infected with the indicated virus at the indicated MOI for 72h (n=3, +/- SD). (B): Same in Vero cells, except rHCoV-OC43/NLuc-NSP3/eGFP was infected for 48h. (C): Flow cytometry analysis of 293T cells infected with rHCoV-OC43/eGFP at MOI 0.1 for 72h, with normal human serum added at 4 hpi (representative experiment, n=3). (D): Same with A549 cells. (E): Flow cytometry analysis of Vero cells infected with rHCoV-OC43/eGFP at MOI 1 for 24h with soluble heparin added concurrently with infection. (F): Vero cells expressing LgBiT were infected with rHCoV-OC43/M-HiBiT at MOI 1 for 24h in the presence of the luciferase detection reagent endurazine. Soluble heparin was added at 0 hpi and/or at 24 hpi, and luminescence detected at 25 hpi (n=3. +/-SD). (G): qPCR of A549 cells infected with HCoV-OC43 at MOI 0.1 for 24h in the presence of Molnupiravir (representative images, n=3). (H): Western blot of EV or RNF213 KO A549 cells infected for the indicated time with HcoV-OC43 and 200 μM Molnupiravir (representative images, n=3.

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Flow Cytometry, Transfection, Infection, Virus, Expressing, Luciferase, Western Blot