Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs

Figures

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  Figure 1. Intracellular SINV RNA levels in BHK cells are increased by higher MOI and by anti-E2 antibody treatment. Time-lapse live-cell imaging (A), objective lens 20X) of TE-UTR-4Br-infected BHK cells (3–5.5 h after infection) on the same settings shows the effects of multiplicity of infection (10 vs. 100) and anti-E2 [SV127] antibody treatment (5 μg/ml, 2 h after infection) on the accumulation of viral RNA (green). The graph (B) shows the average signal intensity of the 5 brightest cells from each group (1 frame = 150 seconds). Scale bar = 100 μm.

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  Figure 2. Effect of anti-E2 antibody treatment on viral RNA in BHK cells. Live-cell imaging (12 h after infection, objective lens 20X) to compare the effect of anti-E2 antibody treatment (5 μg/ml, 2 h after infection) (B) to no treatment (A) on genomic RNA levels and distribution in TEnsP3-4Br-infected BHK cells (MOI = 20). Without antibody treatment (A) the viral genomic RNA (green) is mainly present in the perinuclear area. With antibody treatment the RNA is accumulated at the plasma membrane (B). Scale bar = 100 μm.

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  Figure 3. Antibody- and IFNγ-mediated SINV RNA clearance from dAP7 cells. Live-cell imaging (objective lens 20X, lambda mode) shows the effects of anti-E2 antibody and IFNγ on viral RNA clearance in TE-UTR-4Br-infected dAP7 cells (MOI = 20) at 12, 24, 48 and 72 h after infection (yellow – autofluorescence; green – Broccoli-DFHBI-1T; red – propidium iodide (dead cells)). All treatments (TrkA control antibody 5 μg/ml; anti-E2 5 μg/ml, rat IFNγ 500 U/ml) were applied 2 h after infection. Scale bar = 100 μm.

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  Figure 4. Effects of anti-E2 antibody and IFNγ on viral RNA clearance and cell survival in dAP7 cells. Flow cytometry histograms of the Broccoli-DFHBI-1T signal in dAP7 cells infected with TEds10Br (MOI = 20). (A) The signal intensity of infected cells at 24 h (blue) and 48 h (red) after infection in relationship to auto fluorescence (gray). (B,C) The effects of treatment at 2 h after infection with anti-E2 [SV127] antibody (blue) and/or IFNγ (black/green) in comparison to untreated cells (red) on the numbers of Broccoli-positive cells at 24 h (B) and 48 h (C). (D) Bar chart depicting the percentages of infected dAP7 cells with Broccoli signal greater than autofluorescence at 24 h (black) and 48 h (gray) after treatment with antibody and/or IFNγ. (E) Bar chart showing the effect of treatments on cell survival.

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  Figure 5. Effects of anti-E2 antibody and IFNγ on viral RNA clearance and cell survival in BHK and cAP7 cells. Flow cytometry histograms show the Broccoli-DFHBI-1T signal at 18 h after infection for BHK (A) and cAP7 (B) cells infected with TEds10Br (MOI = 5) and the effects of treatment with anti-E2 [SV127] antibody (blue) and/or IFNγ (green/red) at 2 h after infection compared to untreated cells (dark gray). (C) Bar chart of the numbers of Broccoli-positive BHK (gray) and cAP7 (hatched) cells after treatment with antibody and/or IFNγ. (D) Bar chart showing the effect of treatments on survival of infected BHK and cAP7 cells.

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  Figure 6. Inverse relationship between the amount of intracellular viral RNA and antibody on the cell membrane. Live-cell imaging (objective lens 20X, lambda mode) shows the amount of viral RNA (green) in TE-UTR-4Br-infected dAP7 (MOI = 20, 24 h after infection), treated with 1 μg/ml of SV127-Dylight594 at 2 h after infection. The cells that have antibody (red) bound to their membranes (white arrows) cleared the viral RNA, while the cells that lack antibody on their membrane did not. Scale bar = 50 μm.

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  Figure 7. Lack of E2 envelope glycoprotein on the surface of some SINV-infected cells. dAP7 cells infected with SINV Toto E2-mCherry (MOI = 20), followed by treatment with anti-E2 [SV127, 5 μg/ml] 2 h after infection were fixed with 3.7% formaldehyde at 24 h were stained with anti-SINV nsP3 (white) and anti-mouse IgG (green). The viral E2 glycoprotein (red) is on the membrane of the cell that antibody (green) can bind (white arrows). In contrast, antibody cannot bind another cell that has only intracellular E2 (red arrows). Images are representative of multiple cells in the culture with similar staining. Scale bar = 100 μm.