Fig 1: N protein-assisted nucleic acid dispersion and expression in the co-culture environment(A)The co-culture system consisted of A549 as recipient cells, and 293T pre-transfected with two plasmids, one expressing GFP and the other expressing SARS-CoV-2 N protein or the pcDNA3.1 empty vector.(B–E) After 24 h co-culture of the donor cells and recipient cells, cell pool was stained with cytokeratin 18 (an A549 marker) and SV40 large T antigen (a 293T marker). A549 cells in the cell pool were gated from cytokeratin 18 positive and large T antigen negative. A549 GFP positive percentage was further assessed by flow cytometry analysis. Effects of SARS-CoV-2 N variants (B), treatment with RANTES (C), the p38 inhibitor SB203580 (D), or anti-N neutralizing antibody (E) were accessed by adding these effectors to the medium. Experiments are performed in three to five biological replicates. *, p value <0.05 (paired two-tailed student’s t-test).(F) SARS-CoV-2 N protein promotes gene delivery by cell-free diffusion to neighboring cells. A549 cells were plated in the lower chamber, while 293T donor cells co-transfected with plasmids expressing EGFP and indicated N proteins in the upper chamber. After 3 days of co-culture, GFP positive A549 cells were observed and counted. See also Figures S10 and S11.
Fig 2: Working hypothesisA working hypothesis for SARS-CoV-2 N protein is proposed: Expression of SARS-CoV-2 N protein promotes p38 MAPK activation and RANTES chemokine secretion (1,2). N protein/nucleic acid complexes are secreted (3). The chemokines induced by N protein attract T cells, which produce more RANTES (4). The infected cells may cause local hypoxia and produce lactate (5). RANTES and lactate enhance N-protein-nucleic acid endocytosis by neighboring cells (6,7), and cause more tissue damage. The RNA/DNA carried by N protein could be originated from the virus or the host cell.
Fig 3: Lactate and RANTES enhance N protein-carried nucleic acid expression(A) Lactate effect. 293T cells were pretreated with lactate at indicated concentrations. After treatment, N-GFP DNA mixtures were added to 293T cells. The GFP+ cells were counted after 24 h.(B) Dose-dependent effect of RANTES. 293T cells were treated with an indicated dose of RANTES overnight. After treatment, Pre-mixed 10 µg mammalian cells-expressed WT N or Omicron N and 40 µg GFP-RNA (Left panel), GFP-DNA (Right panel) was added to cells overnight, and the number of GFP+ cells were counted under fluorescent microscopy.(C) N-RNA and N-DNA complexes induced RANTES. HPAEpiC cells were stimulated by mammalian expressed N protein, GFP-RNA (or DNA), and N+GFP-RNA (or DNA) mixture for 24 h. After treatment, protein secretion of RANTES was detected by flow cytometry analysis.(D) N protein was added to HPAEpic cells at indicated dosages for 30 min. After treatment, the cells were harvested and phosphorylation of p38 was detected by Western blotting. Quantification of band intensities was normalized by non-treated cells.(E) 293T cells were pretreated by p38 inhibitor (SB203580) at indicated dosages overnight. After treatment, the N-GFP-DNA mixtures were added to 293T cells. GFP+ cells number were counted after 24 h.(F) Mammalian cells-expressed N protein 4 µg pretreat with anti-N monoclonal antibody (NP-mAb-40 or 56) 10 µg/mL, and then mix with 12 µg GFP-DNA for 1 h, then the N+DNA/antibody complex are added to HPAEPic cells for 48 h. RANTES concentration in the supernatant is measured and detected by anti-RANTES beads by flow cytometry analysis. All data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; t test. See also Figures S6–S9.
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