Journal: PLoS Pathogens

Article Title: Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication

doi: 10.1371/journal.ppat.1008690

Figure Lengend Snippet: (A-C) Vero, H1299, and DF-1 cells were infected with IBV Beaudette strain at an MOI of 1 or mock infected. At the indicated time points, cells were subjected to immunostaining. Infected cells (red) were identified using a rabbit anti-N protein, SGs (green) with a (cross-reacting) mouse anti-G3BP1 and cell nuclei with DAPI (blue). The bar graphs showed the percentage of SGs positive cells to infected cells, which was counted in 20 random fields, presented as mean ± SD. (D and E) Vero and H1299 cells were infected with IBV as described in A-C. At 20 h.p.i., immunostaining was used to visualize the position of the structural components of SGs using anti-G3BP1 (red) and anti-TIAR (green) antibodies. The enlargement of the insets confirms their co-localization in cytoplasmic granules. The representative images of three independent experiments were shown. Scale bars: 10 μm.

Article Snippet: Next, the capped full-length RNA and IBV-N transcripts dissolved in 400 μl PBS were co-transfected into Vero cells by electroporation (450 v, 50 μF, 3 ms, GenePulser Xcell, BIO-RAD).

Techniques: Infection, Immunostaining

Journal: PLoS Pathogens

Article Title: Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication

doi: 10.1371/journal.ppat.1008690

Figure Lengend Snippet: (A and B) Vero and H1299 cells were infected with IBV at a MOI of 1. Mock infection was included as negative control, poly I:C transfection for 6 h or 1 mM sodium arsenite for 30 min were included as positive control. Cells were harvested at the indicated time points and processed for western blot analysis using 10 μg total protein per lane. p-PKR, PKR, p-eIF2α, eIF2α, G3BP1, TIA-1, and IBV-N were detected with corresponding antibodies. β-actin was probed as a loading control. The representative images of two independent experiments were shown. The signals of protein bands were determined by Image J. The intensities of p-PKR or p-eIF2α were normalized to total PKR or total eIF2α. The ratio of p-PKR and p-eIF2α in IBV-infected cells, poly I:C-transfected cells, or sodium arsenite-treated cells, to mock-infected cells, were shown as p-PKR (+:-) or p-eIF2α (+:-).

Article Snippet: Next, the capped full-length RNA and IBV-N transcripts dissolved in 400 μl PBS were co-transfected into Vero cells by electroporation (450 v, 50 μF, 3 ms, GenePulser Xcell, BIO-RAD).

Techniques: Infection, Negative Control, Transfection, Positive Control, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication

doi: 10.1371/journal.ppat.1008690

Figure Lengend Snippet: (A) Schematic diagram of the nsp15 mutation site (H238-A) as described in the Material and methods, and the replication dynamics of IBV and rIBV-nsp15-H238A in Vero, H1299, and DF-1 cells. Cells were infected with IBV or rIBV-nsp15-H238A at an MOI of 1. Cell culture supernatants were collected at the indicated times at 4 h intervals and viral titers were determined by TCID 50 in Vero cells. The dot graphs showed the growth kinetics of IBV and rIBV-nsp15-H238A of three independent determinations, presented as the mean ± SD. (B) Vero, H1299, and DF-1 cells were infected with IBV or rIBV-nsp15-H238A at a MOI of 1 for 20 h, followed by immunostaining. Infected cells were identified with anti-IBN-N (red) and the SGs were detected with anti-G3BP1 (green). Cell nuclei were stained with DAPI (blue). The bar graphs on the right panel showed the percentage of SGs positive cells to total infected cells, which were calculated over 20 random fields, presented as the mean ± SD. P values were calculated by Student’s test. ***, P < 0.001; ****, P < 0.0001. (C) H1299 cells were infected with rIBV-nsp15-H238A for 20 h and treated with 100 μg/ml of cycloheximide (CHX) or an equivalent volume of DMSO for 1 h, followed by immunostaining with anti-G3BP1 or anti-G3BP2 antibodies. The bar graphs showed the percentage of SGs positive cells to total cells, which were calculated over 20 random fields, presented as the mean ± SD. ****, P < 0.0001. The representative images of three independent experiments were shown. Scale bars: 10 μm.

Article Snippet: Next, the capped full-length RNA and IBV-N transcripts dissolved in 400 μl PBS were co-transfected into Vero cells by electroporation (450 v, 50 μF, 3 ms, GenePulser Xcell, BIO-RAD).

Techniques: Mutagenesis, Infection, Cell Culture, Immunostaining, Staining