Journal: bioRxiv
Article Title: SARS-CoV-2 Defective Viral Genomes from Distinct Genomic Regions Drive Divergent Interferon Responses
doi: 10.64898/2026.03.19.712870
Figure Lengend Snippet: (A-C) A549-ACE2 cells were infected with SARS-CoV-2 or DVG-B (both MOI 1) and cells were collected at 6-, 10- and 24-hours post-inoculation for epifluorescence microscopy to ( A ) compare the cellular distribution of SARS-CoV-2 and DVG-B derived dsRNA or ( B ) quantify the total area of dsRNA signal and total dsRNA signal (integrated signal density). Insets depict magnified images of cells indicated by the white boxes. Scale bars indicate 10μm and 25μm, respectively. ****p<0.0001 by two-way ANOVA with Bonferroni’s multiple comparisons test. 6hpi N=4 fields. 10hpi N=5 fields. 24hpi, SARS-CoV-2 N=16 fields, DVG-B N=34 fields. Mean±SD. ( C ) IFNB1 and Spike gene expression were calculated relative to GAPDH by RT-qPCR. IFNB1 copy number values were normalized to mock for each respective timepoint. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA with Turkey’s multiple comparisons test. N=3, mean±SD. ( D-E ) A549-ACE2 cells treated with 10μM remdesivir or an equal volume of DMSO were infected with SARS-CoV-2 or DVG-B (MOI 1) for 24-hours post-inoculation, respectively. N=2 biological repeats ( D ) Epifluorescence images of dsRNA and IRF-3 staining in DMSO (left) and remdesivir (right) treated cells. Scale bars indicate 25μM. Arrowheads indicate dsRNA+ cells with nuclear IRF-3. Remdesivir treated SARS-CoV-2 and DVG-B infections N=3 fields, respectively. DMSO treated SARS-CoV-2 infections N=14 fields, DVG-B infections N=30 fields. ( E ) Quantification of the proportion of dsRNA+ cells with nuclear IRF-3 relative to the total number of dsRNA+ cells per field. Each point represents one field. ****p<0.0001 by unpaired two-tailed t-test. SARS-CoV-2: 412 dsRNA+ cells among N=14 fields. DVG-B: 120 dsRNA+ cells among N=30 fields. ( F ) A549-ACE2 cells treated with 10μM remdesivir or an equal volume of DMSO were infected with SARS-CoV-2 or DVG-B (both MOI 1) or were transfected with low molecular weight poly(I:C) (PIC) for 24-hours post-inoculation, respectively. IL-29 and Spike gene copy numbers were calculated relative to GAPDH by RT-qPCR. **p<0.01, ****p<0.0001 by mixed-effects analysis with Bonferroni’s multiple comparisons test. N=3, mean±SD. ( G ) A549-ACE2 cells were treated with 10μM remdesivir or an equal volume of DMSO and infected with DVG-A (MOI 1) for 24-hours post-inoculation. DVG-A levels were quantified relative to GAPDH by RT-qPCR. N=4, mean±SD. ( H ) A549-ACE2 MAVS KO, PKR KO, or empty vector transduced control cells (parent) were infected with DVG-B (MOI 5) for 24-hours post-inoculation. IFNB1, IL-29, ISG54 and DVG-B copy numbers were quantified relative to GAPDH by RT-qPCR. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. N=4, mean±SD.
Article Snippet: Alternatively, cells were transfected with 1 μg/mL low molecular weight poly(I:C) (InvivoGen catalog no. tlrl-picw) via lipofectamine 2000.
Techniques: Infection, Epifluorescence Microscopy, Derivative Assay, Gene Expression, Quantitative RT-PCR, Staining, Two Tailed Test, Transfection, Molecular Weight, Plasmid Preparation, Control
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