Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

Figures

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  Figure 1. Sites for insertion and production of the SINV-Broccoli fluorescent signal. (A) Sequence of the 3′end of SINV TE or TEds showing sites of F30-2xdBroccoli (234 bases) insertion at the SpeI site in nsP3 (not shown) and/or at the BstEII site (green characters) downstream of the second subgenomic promoter (bold characters) and/or at the SphI site in the 3′UTR created by site-directed mutagenesis (blue characters). (B) Example of a constructed viral RNA genome with F30-2xdBroccoli inserted in the 3′UTR inside infected cells. Four copies of Broccoli RNA aptamers bind the cell-permeable fluorophore in the medium and emit green fluorescence upon blue laser excitation.

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  Figure 2. F30-2xdBroccoli and construction of tandem repeats. (A) Sequence of F30-2xdBroccoli with 2 copies of Broccoli dimers (bold green characters) in the F30 scaffold (black characters). (B) Tandem repeats consist of 3 sets of F30-2xdBroccoli (12 Broccoli in total), made from self-ligation between the complementary 5′ (BamHI – G^GATC_C) and 3′ end (BglII – A^GATC_T). The ligation forms a scar (GGATCT), which can no longer be cut by either BamHI or BglII. The tandem repeats can be inserted into 3 restriction sites of TEds plasmid in the specific order – nsP3, 3′UTR, and downstream of 2nd subgenomic promoter.

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  Figure 3. Construction of multiple SINV-Broccoli recombinant viruses. 1–3 repeats of F30-2xdBroccoli were inserted into 3 restriction sites: SpeI in nsP3, BstEII downstream of the 2nd subgenomic promoter, and SphI in the 3′UTR.

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  Figure 4. Comparison of signal intensities of all Spinach2 and Broccoli aptamer-tagged SINVs. The live cell images (objective lens 20×) compare signal intensities of all Spinach2 (Sp) and Broccoli (Br) recombinant viruses after adding DFHBI-1T to the infected BHK cells (MOI = 5, 6 h after infection). Scale bar = 100 μm.

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  Figure 5. The effect of formaldehyde fixation on the Broccoli signal. The images (objective lens 20×) show BHK cells infected (MOI = 5) with TE (upper) or TEds6Br (lower) at 16 h after infection. Panels on the left are live cell images with and without addition of DFHBI-1T. Shortly after imaging, the same cells were fixed with 3.7% formaldehyde and re-imaged (panels on the right). Scale bar = 100 μm.

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  Figure 6. Colocalization between SINV RNA FISH and Broccoli. The images (objective lens 20×) show infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS 6 h after infection. After adding DFHBI-1T, RNA FISH E1 + E2 (red) colocalizes well with Broccoli-DFHBI-1T (green) signal for both TEUTR4Br and TEds10Br. The scatter plot (X – red, Y – green) shows strong colocalization (Rcolocalization = 0.9049). Scale bar = 100 μm.

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  Figure 7. Rare discrepancy between RNA FISH and Broccoli signals. The images (objective lens 63×) show TEds10Br-infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS at 6 h after infection. Although rare, some cells showed a discrepancy between FISH and Broccoli signals (white arrow), resulting in a separated cluster in the scatter plot (X – red, Y – green). Scale bar = 20 μm.

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  Figure 8. Replication of SINV-Broccoli in BHK and AP7 cells and in mouse brain. The graphs show the amount of infectious virus in supernatant fluids from BHK (A), cAP7 (B), or dAP7 (C) cells infected (MOI = 5) with SINV-Broccoli recombinant viruses (3 replicates per virus per time point). *P < 0.05; **P < 0.01; ***P < 0.001 (2-way ANOVA). Limit of detection was 50 pfu/ml (log10 1.7). (D) Amount of infectious virus in brain homogenates from 4–6 week-old C57BL/6 mice 3 days after intracerebral infection with 1000 pfu SINV-Broccoli recombinant viruses (3–6 mice per virus).

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  Figure 9. Accumulation of viral RNA in the foot processes of differentiated neurons. Live cell imaging (objective lens 20×) in lambda mode (spectral imaging) of TE (control) and TEds10Br-infected dAP7 cells (MOI = 20, 24 h after infection) plus DFHBI-1T. Viral RNA (green – Broccoli-DFHBI-1T) accumulated at filopodial tips (red arrow heads) in contact with an uninfected cell (yellow autofluorescence). Scale bar = 100 μm.

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  Figure 10. Live cell imaging of infected cells in mouse brain slices incubated with DFHBI-1T. (A) Channel mode and (B) lambda mode imaging of fresh TEds10Br-infected brain or (C) TE-infected brain. Broccoli-DFHBI-1T signal (green) in cells of the corpus callosum. (D) RNA E1 FISH imaging (red) of infected neurons in formalin-fixed brain tissue counterstained with DAPI. (E) Image of the 250 μm slice (McIlwain tissue chopper) of TEds10Br-infected mouse brain (3 days after infection) in a 35-mm glass-bottom dish in imaging medium containing DFHBI-1T with area of imaging for A, B indicated in the red square. Scale bar = 50 μm.

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  Figure 11. Distribution of SINV genomic and subgenomic RNA in BHK cells. (A) Live cell imaging (objective lens 63×) of TEds6Br-infected BHK cells (MOI = 5, 16 h after infection) shows more diffuse distribution of viral RNA (green – Broccoli-DFHBI-1T) when both genomic and subgenomic viral RNAs are labeled with Broccoli. (B) Cells infected with TEnsP3-4Br with labeled genomic RNA show focal accumulations of RNA in the perinuclear area. (C) Control image of uninfected cells incubated with DFHBI-1T. Scale bar = 20 μm.

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  Figure 12. Vesicle-like arrangement of viral RNA. Live cell imaging of TEds14Br-infected BHK cells (MOI = 5, 20 h after infection) shows perinuclear vesicle-like arrangement of viral RNA (green – Broccoli-DFHBI-1T) in orthogonal view (A,B – zoom in). (C) TE-infected cells incubated with DFHBI-1T. Scale bar = 20 μm (A,C); 5 μm (B).