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. 2023 May 24;26(6):106954. doi: 10.1016/j.isci.2023.106954

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© 2023 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Analysis of spike protein function and creation of COVID-19 encephalopathy model using S1 protein

(A) Structures of genes used in expression experiments. SS, signal sequence; S1/S2, S1/S2 protease cleavage site; FP, fusion peptide; HR1, heptad repeat 1; R2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail.

(B) Comparison of intracellular calcium concentration elevation among vector control, spike protein, S1 protein and NTD in mouse 3T3 cells. Calcium concentration was measured by fluorescence of Fluo-4. Vector control, n = 750; spike protein, n = 750; S1 protein, n = 746; NTD, n = 740; Kruskal-Wallis test followed by Dunn’s post hoc test; median values; ∗∗∗∗, p < 0.0001.

(C) Measurement of intracellular calcium concentration in human A549 cells. Calcium concentration was measured by fluorescence of Fluo-4. Vector control, n = 444; S1 Adv, n = 477; Mann-Whitney U-test; median values; ∗∗∗∗, p < 0.0001.

(D) S1 mouse creation and evaluation.

(E) Measurement of apoptosis induction in S1 mouse olfactory bulb tissue based on caspase 3 positive cell count (vector control, n = 5; S1 mouse, n = 5; Mann-Whitney U-test; median values; ∗∗, p < 0.01).

(F) Reduced swimming time in S1 mouse WFST; i.e., increase in fatigue (vector control, n = 20; S1 mouse, n = 20; Mann-Whitney U-test; median values; ∗∗∗∗, p < 0.0001).

(G) Prolonged immobility time in S1 mouse TST, i.e., increase in depressive clinical signs (vector control, n = 11; S1 mouse, n = 12; Mann-Whitney U-test; median values; ∗, p < 0.05).

(H) Enhanced IL-6 expression in S1 mouse brain tissue (vector control, n = 5; S1 mouse, n = 5; Mann-Whitney U-test; median values; ∗, p < 0.05).

(I) Enhanced IL-6 expression in S1 mouse lung tissue (vector control, n = 10; S1 mouse, n = 10; Mann-Whitney U-test; median values; ∗∗, p < 0.01).

(J) S1 mRNA expression in lung, olfactory bulb and brain tissue (Ratios of S1 to 18s ribosomal RNA are shown.) (vector control, n = 4 each; S1 mouse, n = 4 each median values).

(K) Comparison of intracellular calcium concentration elevation among Wu strain, α strain, and δ strain in mouse 3T3 cells. Calcium concentration was measured by fluorescence of Fluo-4. Vector control, n = 3562; Wu strain, n = 2711; α strain, n = 2837; δ strain, n = 2339; Kruskal-Wallis test followed by Dunn’s post hoc test; median values; ∗∗∗∗, p < 0.0001.