Nuclear translocation of spike mRNA and protein is a novel feature of SARS-CoV-2

Figure 1. Only the SARS-CoV-2 S protein had an NLS motif “PRRARSV” due to a novel sequence insertion. (A) Full-length SARS-CoV-2 genome (nucleotide) (USA/WA-CDC-WA1/2020 isolate, GenBank accession no. MN985325) and open reading frames (ORF) are shown at the top. The SARS-CoV-2 S protein amino acid sequence was aligned with SARS-CoV (Urbani strain, GenBank accession no. AY278741) by NCBI’s constraint-based multiple alignment tool COBALT (Papadopoulos and Agarwala, 2007), and the relative positions of four novel sequence insertions (ISs) are shown in the S protein ORF as follows: IS1: “GTNGKTR,” IS2: “YYHK,” IS3: “HRSY,” and IS4: “NSPR.” The fourth IS (NSPR) created a pat7 NLS “PRRARSV” in the S protein (shown in the red rectangle). (B) The S protein ORF sequences between SARS-CoV-2 and SARS-CoV were aligned, and the Lined rectangles highlight the four novel insertions: IS1, IS2, IS3, and IS4. The IS4 “NSPR” created a pat7 NLS “PRRARSV” in the S protein (shown in the black rectangle).

Figure 2. The intracellular distribution of S mRNA and S protein suggests nuclear translocation. Four-week highly differentiated pseudostratified airway epithelium was infected with SARS-CoV-2 at a MOI of 0.1 for 4 days, paraformaldehyde-fixed, paraffin-embedded, and sectioned at a thickness of 5 μm for immunohistochemistry (IHC) and slide preparation (Osan et al., 2020, 2021). A combined protocol of RNAscope and IHC was used to simultaneously detect S mRNA and S protein in the SARS-CoV-2-infected airway epithelium. S mRNA (red) was detected using a SARS-CoV-2 S mRNA probe for RNAscope, and S protein (cyan) was detected by an S protein-specific rabbit polyclonal antibody and a corresponding anti-rabbit secondary antibody for immunofluorescence (IFA) analysis. The nucleus (blue) was detected by DAPI staining. The images were taken under an Olympus confocal microscope using a 60x oil objective. The images represent multiple independent technical replicates from two independent experiments with different donors (experiment 1: donors 2 and 3 and experiment 2: donor 1). The scale bar is 10 μm.

Figure 3. The nuclear translocation of S protein and S mRNA includes both the outer surface and inside of the nucleus. Separate slides (see Figure 2) were imaged under a Leica Stellaris confocal microscope (Leica) using a 63x oil objective. The images were then deconvolved using Huygen Essential deconvolution software (Scientific Volume Imaging). Using the surface rendering function of an image processing IMARIS software. (A) S mRNA (red) on the nuclear surface (top) and inside the nucleus (bottom). White arrows indicate S protein on the nuclear surface (top image) or inside the nucleus (bottom image). (B) S protein (green) on the nuclear surface (top image) and inside the nucleus (bottom image). White arrows indicate S protein on the nuclear surface (top image) or inside the nucleus (bottom image). (C) The total distribution of S mRNA and S protein in the cells. The data were obtained by combining multiple images from an independent experiment. (D) The total colocalization between S mRNA and S protein in the cells. The data were obtained by combining multiple images from an independent experiment.

Figure 4. Colocalization between S mRNA and S protein inside infected cells. The images (see Figure 3) were analyzed by using the surface rendering and colocalization features of IMARIS. S protein and S mRNA distribution and colocalization in the cytoplasm (top panel), on the nuclear surface (middle panel) and inside the nucleus (bottom panel). The specific region of colocalization is indicated by a white spot. Scale bar 0.5 μm.

Figure 5. S protein in both cytoplasmic and nuclear fraction of the transfected A549 cells. A549 cells were transfected with SARS-CoV-2 S plasmid (Leventhal et al., 2021) for 72 h. SARS-CoV-2 S protein was detected in both cytoplasmic and nuclear fractions by Western blotting. Cdc-42 detection confirms cytoplasmic portion and Lamin A and C detection confirms nuclear fraction.

Figure 6. The nucleoproteins of SARS-CoV, MERS-CoV, and SARS-CoV-2 translocate into the nucleus. (A) Four-week pseudostratified airway epithelium was infected with SARS-CoV-2 at a MOI of 0.1 for 4 days, fixed, paraffin-embedded, and sectioned at a thickness of 5 μm for immunohistochemistry slide preparation. Simultaneous detection of S mRNA (shown in red) and N protein (shown in cyan) on the same slide was performed by a combined detection protocol in RNAscope-based mRNA and immunofluorescence-based protein detection. An S mRNA-specific probe was used for RNAscope, and an N protein-specific rabbit polyclonal antibody and the corresponding anti-rabbit secondary antibody were used. The nucleus (shown in blue) was detected by DAPI staining. The images were taken under an Olympus confocal microscope using a 60x oil objective. The images represent multiple independent technical replicates from two independent, healthy donors (top row: donor #1 and bottom row: donor #2). The scale bar is 10 μm. (B) Four-week pseudostratified airway epithelium was infected with SARS-CoV-2, SARS-CoV, or MERS-CoV at an MOI of 0.1 for 4 days. SARS-CoV-2 or SARS-CoV N protein was detected by an N protein-specific…