Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence

Figures

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  Figure 1. Design and optimization of SARS-CoV-2-specific smRNA-FISH probes to detect SARS-CoV-2 infection. (A) Schematic representation of SARS-CoV-2 gRNA and sgRNA-S, indicating the position of the smRNA-FISH probes P1 (in magenta) and P2 (in green) that hybridize to spike and nsp12 ORF sequences, respectively. (B) Heatmap representing the probe sequence alignment against various coronavirus genomes (including SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-NL63, and HCoV-229E) and human transcriptomes (hg38-mrna). Each column represents individual 22 nt spike gene probe sequences from the spike gene (S1–S40, upper panel, represented in the shades of red) and from the nsp12 gene (N1–N40, middle panel, represented in shades of green). The lower panel represents the heatmap of the spike probe sequence alignment against the spike gene of SARS-CoV-2 and the codon-optimized spike gene of SARS-CoV-2 (S1–S40, lower panel, represented in the shades of red), showing the specificity of the probe. The minimum edit distance represents the mismatch score between the SARS-CoV-2 sequence and the other genome sequences, where 0 indicates a perfect match (in white) and > 10 represents the most mismatch…

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  Figure 2. Time-course analysis of SARS-CoV-2 replication using smRNA-FISH. Cells were infected and hybridized with Quasar 570-labeled P1 probes (magenta) against spike RNA at 0, 0.5, 2, 6, 12, and 24 h p.i. (A) Photomicrographs representing the images acquired using high-resolution, high-speed scanning microscopy are represented at 2× (upper panels) and 40× (middle panels) magnifications, respectively. Scale bars, 1000 μm and 50 μm for 2× and 40× images, respectively. Graphical representation showing the quantitation of the total percentage of positive cells infected with SARS-CoV-2 at 0, 6, 12, and 24 h p.i. (lower panel). (B) Photomicrographs representing images of infected cells at single-molecule resolution probed with P1 at 0.5, 2, 6, 12, and 24 h p.i., acquired using a wide-field Olympus BX-63 microscope. White arrows point to single molecules of SARS-CoV-2 RNA seen at early time points, 0.5 and 2 h p.i. Yellow arrow heads at 6 h p.i. point to RNA clusters/speckles. DAPI (nucleus, blue). Scale bar, 10 μm.

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  Figure 3. Simultaneous detection of SARS-CoV-2 gRNA and sgRNA-S in a time-course analysis. (A) Validation of spike RNA probe P1 and nsp12 RNA probe P2 to simultaneously detect gRNA and sgRNA-S in the infected cells. Cells were infected for 6 h and hybridized with probes P1 and P2 alone or together and subjected to smRNA-FISH. Images are represented at 20× magnification. Scale bar at 100 μm. (B) A time-course analysis to detect the replication of gRNA and sgRNA in infected cells: The infected cells at 2, 3, 4, and 5 h p.i. were hybridized with both spike RNA probe P1 and nsp12 RNA probe P2 and subjected to high-speed, high-resolution scanning. The images are represented at 76.3× magnifications. Panels 4, 8, 12, 16, and 20 are the zoomed-in images of insets for the indicated time points. White arrows point to RNA spots that are surrounded by potentially single-molecule sgRNA-S. Blue indicates DAPI-stained nuclei. Magenta indicates sgRNA-S, green indicates gRNA and white color indicates the overlap of the two probes. Scale bar at 20 μm.

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  Figure 4. Single-molecule analysis to determine the kinetics of SARS-CoV-2 gRNA and sgRNA replication. Transcript-specific visualization of gRNA and sgRNA-S in SARS-CoV-2-infected Vero E6 cells using the P1 (spike, represented in magenta) and P2 (nsp12, represented in green) probes. When spike RNA probe P1 and nsp12 RNA probe P2 both hybridized to the same molecule, it is shown in white (overlay). Blue indicates DAPI-stained nuclei. Images represent the cells infected with SARS-CoV-2 and hybridized with the P1 and the P2 probes at 0, 2, 3, 4, 5, and 12 h p.i. The white arrows in panels 6–16 point to replication centers. The yellow arrowheads point to single molecules of sgRNA-S. Panels 13–16 show the magnified area of the corresponding overlay images at 3 h p.i., illustrating two replication centers (white arrows). The larger RNA spots in these panels harbor gRNA in the center surrounded by sgRNA-S. Single-molecule RNA are indicated by yellow arrowheads (panels 14–16). The white arrows in panels 22–24 point to RNA in the nucleus. The images were acquired using a wide-field microscope to detect the diffraction-limited single molecules. Scale bar at 10 μm.

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  Figure 5. Images of cells infected with SARS-CoV-2 at 5 h p.i. (A) Representative cells infected with SARS-CoV-2 at 5 h p.i. Panels 1–4 represent a cell filled with ROs at 5 h p.i. Panels 5–8 represent a single cell with gRNA and sgRNA-S migrating to the distal end of the cell (yellow arrowheads). Yellow dotted line in panel 8 represents the outline of the cell. (B) Presence of SARS-CoV-2 RNA in the nucleus. The images from a few Z-stacks of the cell are represented to demonstrate the presence of the positive RNA spot inside the nucleus. The Z-stack images were acquired starting from the bottom of the cell moving upwards. The panel numbers represent the stack number in a total of 41 optical sections (each with a 200 nm Z step size). In both A and B, images were acquired using a wide-field microscope to detect the diffraction-limited single molecules. The spike RNA P1 probe signals are colored in magenta (sgRNA-S), nsp12 RNA P2 probe signals are colored in green (gRNA), and when P1 and P2 both hybridized to the same molecule, it is shown in white (overlay). Scale bar, 10 μm. DAPI (nucleus, blue).

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  Figure 6. Co-localization of SARS-CoV-2 RNA speckles with nsp3 protein. Representative cells showing the co-localization of spike probe P1 and nsp3 protein within the Vero E6 cells infected with SARS-CoV-2 at 3, 6, and 12 h p.i., respectively. The images were acquired using a wide-field microscope to detect the diffraction-limited single molecules. The blue color represents DAPI nuclear staining, the green color represents nsp3 protein, and the magenta color represents SARS-CoV-2 spike RNA. Scale bar, 10 μm. Panels 21–24 show the magnified area of the corresponding images at 3 h p.i. (scale bar at 2 μm). White arrows indicate RNA speckles that co-localized with nsp3. Black arrow point to the same panel as in panel 4, whose blown up images are shown in panels 21–24.

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  Figure 7. Quantitation of cells containing viral RNA at different stages of replication. (A) Examples of images of cells with four different stages of viral replication at stage 1 (panels 1 and 2), stage 2 (panels 3 and 4), stage 3 (panels 5 and 6), and stage 4 (panels 7 and 8). The blue color represents DAPI nuclear staining, and nsp12 RNA (P2 probe) signals are colored in green, which represents replication centers containing genomic RNA. Scale bar, 50 μm. (B) Graphical representation of the quantitation of the percentage of positive cells harboring four different stages of replication at 4, 5, and 6 h p.i.

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  Figure 8. Ability of SARS-CoV-2 probes to detect variants of concern (VOCs) of SARS-CoV-2. (A) Heatmap representing the probe sequence alignment against various genomes of SARS-CoV-2 variants of concern (VOCs) (SARS-CoV-2 B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529, BA.1, BA.2, BA.4, and BA.5) along with SARS-CoV-2 Wuhan-Hu-1. Each column represents individual 22 nt spike gene probe sequences from the spike gene (S1–S40, upper panel, represented in the shades of red) and from the nsp12 gene (N1–N40, lower panel, represented in shades of green). The minimum edit distance represents the mismatch score between the SARS-CoV-2 Wuhan-Hu-1 gene sequence and the other SARS-CoV-2 VOC genome sequences, where 0 indicates a perfect match (in white) and > 10 represents the most mismatch (in dark red or green). (B,C) Validation of spike RNA probe P1 and nsp12 RNA probe P2 to simultaneously detect gRNA and sgRNA-S in the Vero-TMPRSS2 cells infected with SARS-CoV-2 BA.1 VOC. Cells were infected at 3 h, 6 h, and 12 h. (B). Panels 2, 4, and 6 indicate infected cells and panels 1, 3, and 5, represent mock-infected cells, shown at a 20× magnification. Panel 7 indicates Vero-TMPRSS2 cells infected with…