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

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: Overexpression of SERINC1, 3, and 5 impairs infectivity of hCoV‐OC43 but not hCoV‐229E. (A and B) Huh7 cells transiently transfected with empty or SERINC encoding pBJ6 vectors were infected with hCoV‐229E (A) or hCoV‐OC43 (B) at the indicated MOIs. At 30 hpi (A) or 48 hpi (B), supernatants and cell lysates were collected for qRT‐PCR and western blot analysis, respectively. (C) Supernatants of Huh7 cells treated as described in (A) were harvested for TCID 50 determination at 30 h postinfection. (D) Huh7 cells were treated as described in (B) and supernatants harvested for TCID 50 assay at 72 h postinfection. Shown are mean values ± SEM from three or four independent experiments. Statistical significance compared with the mock control was assessed using unpaired t ‐test with Welch's correction. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Over Expression, Infection, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: Endogenous SERINC1, 3, 4, and 5 restrict infectivity of hCoV‐OC43 but not hCoV‐229E. (A) Huh7 cells transfected with nontargeting or SERINC‐specific siRNA were infected with hCoV‐229E at an MOI of 0.001 or hCoV‐OC43 at MOI 0.01. Supernatants were harvested at 30 or 72 hpi for qRT‐PCR and TCID 50 . Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. (B) NHLF cells transfected with nontargeting or SERINC‐specific siRNAs were infected with hCoV‐229E (MOI 0.02)) or hCoV‐OC43 (MOI 0.002). Supernatants were collected at 36 hpi (hCoV‐229E) or 72 hpi (hCoV‐OC43) for qRT‐PCR and TCID 50 . Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. (C) Supernatants from (B) were used to infect A549 cells. Viral infection was assessed by immunofluorescent staining of the hCoV‐OC43 nucleocapsid (purple) and DAPI‐stained nuclei (blue). Scale bar: 40 µm. Infection rates were quantified using image J. A total of 28 images (seven images from each independent experiment) per condition were included for analysis. Shown are mean values ± SEM from three or four independent experiments. Statistical comparisons with mock control were performed using unpaired t ‐test with Welch's correction. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Infection, Transfection, Quantitative RT-PCR, Plasmid Preparation, Control, Staining

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: hCoV‐229E and hCoV‐OC43 reduce exogenous SERINC expression levels. (A) Huh7 cells transiently transfected with HA‐tagged SERINCs were infected with hCoV‐229E (MOI 0.001) or hCoV‐OC43 (MOI 0.01). Whole cell lysates and supernatants were harvested at 3 days postinfection (dpi) for western blot. Shown are the western blot analyses of cell lysates and purified virions representative for four independent experiments. (B) Huh7 cells transfected with GFP‐tagged SERINC1, SERINC2, SERINC3, and SERINC5 were infected with hCoV‐229E (MOI 0.01), hCoV‐OC43 (MOI 0.1), or left untreated. Immunofluorescence staining was performed at 2 dpi. Nucleocapsid proteins (purple) of hCoV‐229E or hCoV‐OC43 were stained to indicate infection; nuclei were stained with DAPI (blue). Scale bar: 20 µm. Results are representative of at least three independent experiments.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Expressing, Transfection, Infection, Western Blot, Purification, Immunofluorescence, Staining

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: SERINC5 degradation by hCoV‐229E infection is lysosome dependent. (A) Huh7 cells transiently transfected with GFP‐SERINC5 were infected with hCoV‐229E (MOI 0.01), hCoV‐OC43 (MOI 0.1), or left untreated. Immunofluorescence staining was performed at 2 dpi. Viral infection was detected via nucleocapsid staining (purple), and lysosomes were marked with LAMP1 (yellow). Colocalization of GFP‐SERINC5 with LAMP1 was quantified using Huygens software ( n = 22 cells). Scale bar: 20 µm. (B) Huh7 cells transiently transfected with vectors expressing GFP alone or GFP‐SERINC5 were infected with hCoV‐229E or left untreated. Mean GFP fluorescence intensity in live cells was measured by flow cytometry at 2 dpi. (C and D) Huh7 cells transfected with GFP‐SERINC5 were infected with hCoV‐229E (C), hCoV‐OC43 (D), or left uninfected, followed by treatment with proteasome inhibitor MG132 (20 µM, 6 h) or lysosome inhibitor NH 4 Cl (10 mM, 18 h). Cells were harvested at 48 h postinfection for flow cytometry analysis. (E) Huh7 cells transiently transfected GFP‐SERINC5 were infected with hCoV‐229E, followed by MG132 treatment for 6 h or NH 4 Cl treatment for 18 h. At 48 hpi, cells were fixed for immunofluorescence staining or harvested for western blot. Scale bar: 20 µm. A total of 45 cells were analyzed for colocalization of GFP‐SERINC5 and LAMP1 using Huygens. Shown are mean values ± SEM from three or four independent experiments. Statistical significance compared with the mock control was assessed using unpaired t ‐test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01. *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Infection, Transfection, Immunofluorescence, Staining, Software, Expressing, Fluorescence, Flow Cytometry, Western Blot, Control

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: hCoV‐229E ORF4a mediates SERINC5 relocalization and degradation. (A) Huh7 cells were cotransfected with constructs expressing GFP‐SERINC5 and the indicated hCoV‐229E proteins. At 2 dpi, cells were analyzed by flow cytometry staining or western blot. Shown are mean GFP fluorescence intensities in live cells, normalized to vector control. Data represent two biological replicates from four independent experiments. (B and C) Colocalization analysis of GFP‐SERINC5 with LAMP1 or with Strep II‐tagged viral proteins in Huh7 cells. A total of 10 to 22 cells in each group were included for analysis. Statistical significance compared with the mock control was determined using unpaired t ‐test with Welch's correction. ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Construct, Expressing, Flow Cytometry, Staining, Western Blot, Fluorescence, Plasmid Preparation, Control

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: hCoV‐229E ORF4 is critical for SERINC5 resistance of hCoV‐229E. (A) Huh7 cells were transfected with a construct expressing SERINC5‐iFLAG followed by infection with wild‐type or ORF4‐deleted GFP‐hCoV‐229E. At 2 dpi, cells were fixed for immunofluorescence staining. SERINC5‐iFLAG was detected via anti‐FLAG staining (purple); LAMP1 (yellow) marks lysosomes; DAPI (blue) stains nuclei. Scale bar: 20 µm. Colocalization of GFP‐SERINC5 with LAMP1 was quantified using Huygens ( n = 21 cells per condition). (B) Huh7 cells transiently transfected with empty or SERINC5 encoding vector were infected with wild‐type or GFP hCoV‐229E at indicated MOIs. At 36 hpi, supernatants and cell lysates were harvested for qRT‐PCR, TCID 50 , and western blot. Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. (C) Huh7 cells transfected with nontargeting or siRNA or SERINC5‐targeting siRNAs were infected with wild‐type or GFP hCoV‐229E at the indicated MOIs. Supernatants were harvested for qRT‐PCR and TCID 50 at 36 hpi. (D) NHLF transfected with nontargeting or SERINC5‐targeting siRNAs were infected with wild‐type or GFP hCoV‐229E (MOI 0.02). Supernatants were collected at 48 hpi for qRT‐PCR and TCID 50 . Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. Shown are mean values ± SEM from three to eight independent experiments. Statistical significance compared with the mock control was determined using unpaired t ‐test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Transfection, Construct, Expressing, Infection, Immunofluorescence, Staining, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control

Journal: bioRxiv

Article Title: Resolving in vitro heterogeneity of host functional responses to HCoV-229E via single-cell analyses

doi: 10.64898/2026.04.17.719293

Figure Lengend Snippet: A) Experimental protocol with sample collection at 24 and 48 hours post infection (hpi) for two duplicate experiments (A and B). For scRNA-seq, this resulted in transcriptomes from 13,913 cells across all samples, including 4,192 mock-infected and 2,724 virus-infected cells at 24 hpi and 3,593 mock-infected and 3,404 virus-infected cells at 48 hpi. B) The viral load across samples after quality control. The median log1p(viral reads) detected in 24 hpi sample A is 7.0 (SD 1.0), in 24 hpi sample B is 7.1 (SD 0.9), and across samples A and B is 7.1 (SD 1.0). The median log1p(viral reads) detected in 48 hpi sample A is 6.6 (SD 1.0), in 48 hpi sample B is 6.4 (SD 1.0), and across samples A and B is 6.5 (SD 1.0). C) Venn diagram representing the number of differentially expressed genes in a cell-level, time-matched 229E-infected versus mock-control differential expression test at a threshold of adjusted p-value < 0.001 and |log ₂ FC| > 2.

Article Snippet: Human coronavirus strain 229E (HCoV-229E) from ATCC ((#VR-740, Lot #70053995) was used for in-house virus batch propagation.

Techniques: Infection, Virus, Control, Quantitative Proteomics

Journal: bioRxiv

Article Title: Resolving in vitro heterogeneity of host functional responses to HCoV-229E via single-cell analyses

doi: 10.64898/2026.04.17.719293

Figure Lengend Snippet: A) The median aggregated accessibility profiles centered on the transcription start sites (TSS; ±3kb) for mock and 229E-infected samples at 24 and 48 hpi. The bold lines show median and mean profiles computed over mean profiles across all samples, while lower opacity profiles show variability across replicates. B) The mean aggregated accessibility profiles centered on the transcription start sites (TSS; ±3kb) for mock and 229E-infected samples at 24 and 48 hpi. The bold lines show median and mean profiles computed over mean profiles across all samples, while lower opacity profiles show variability across replicates. C) Principal component analysis (PCA) of accessibility profiles separates mock-control and 229E infected samples at 24 and 48 hpi. Axes indicate the variance explained by each principal component, marker shapes indicate time point, and marker colors indicate infection status. D) Volcano plots of differential accessibility (infected versus mock-control) at 24 (left) and 48 (right) hpi. Each point is an open chromatin region, and blue points indicate significantly increased accessibility, while orange points indicate significantly decreased accessibility. Gray points are not significant. At 24 hpi, 6% of open chromatin regions (OCRs) show increased chromatin accessibility (blue) and 5% show decreased chromatin accessibility (orange). At 48 hpi, 20% of OCRs show increased chromatin accessibility (blue), and 13% show decreased chromatin accessibility (orange).

Article Snippet: Human coronavirus strain 229E (HCoV-229E) from ATCC ((#VR-740, Lot #70053995) was used for in-house virus batch propagation.

Techniques: Infection, Control, Marker