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-B) A549:EV and A549:N cells were infected with WT (MOI 1) or DVG-B (MOI 1), respectively, and cells were collected 24-hours post-inoculation for confocal microscopy. (A-B) Maximum intensity projections of merged dsRNA ( A-B , red) staining with the N protein ( A , white) or Nsp6 ( B , white) in A549-EV and A549:N cells. Nuclei were Hoechst stained (blue). Note that DVG-B encoded GFP is depicted (green). Scale bar indicates 20μm. Insets depict magnified cell regions indicated by the white boxes of dsRNA and N protein co-staining ( A ) or dsRNA and Nsp6 co-staining ( B ). Scale bar indicates 5μm. Graphs indicate the Pearson’s correlation coefficient ( R) values for the co-localization of dsRNA and N ( C, above) or dsRNA and Nsp6 ( C, below ) in A549:EV and A549:N cells, respectively. Individual values are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Turkey’s multiple comparisons test. Mean±SD. ( D ) A549:EV (left) and A549:N cells (right) were infected with WT (MOI 1) or DVG-B (MOI 50 to maximize the DVG-B infected cell number), respectively, and cells were collected 24-hours post-inoculation for fixation and processing for transmission electron microscopy. Scale bars indicate 2μm. Insets depict magnified cell regions indicated by the white boxes. Scale bars indicate 500nm. Black asterisks indicate single membrane vesicles, red asterisks indicate lysosome-like structures. ( E ) Due to significant more cell death in WT infected A549:EV and A549:N cells, we reduced MOI of WT virus to 0.1 for this experiment. A549:EV and A549:N cells were infected with WT (MOI 0.1) or DVG-B (MOI 1), respectively, and cells were collected 24-hours post-inoculation for RT-qPCR. Expression of host genes and viral genes/DVG-B were calculated relative to GAPDH. IFNB1, IL-29 and ISG54 relative copy number values were normalized to mock for A549:EV and A549:N, respectively. Because the A549:N cell line expresses codon-optimized N, the qPCR primers specifically detect virus-derived N transcripts. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Turkey’s multiple comparisons test. N=2 biological repeats, mean±SD. ( F ) A549:EV and A549:N cells were inoculated as in ( E ). Immediately following 2-hours of inoculation, 10μg/mL of a neutralizing SARS-CoV-2 spike antibody or 10μg/mL of an IgG isotype control antibody were added to the infected cell cultures. 24-hours post inoculation cell supernatants were collected for virus titer determination by TCID 50 /mL quantification. Cells were collected for RT-qPCR. Expression of host genes and viral genes/DVG-B were calculated relative to GAPDH. IL-29 relative copy number values were normalized to mock for A549:EV and A549:N, respectively. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Turkey’s multiple comparisons test. N=3 biological repeats, mean±SD.

Article Snippet: Post inoculation, cells were washed three times with PBS and cultured in 5% TCM with 10μg/mL of Spike neutralizing antibody (40592-R001, Sino Biological, obtained from BEI resources).

Techniques: Infection, Confocal Microscopy, Staining, Transmission Assay, Electron Microscopy, Membrane, Virus, Quantitative RT-PCR, Expressing, Derivative Assay, Control