Fig. 1. The effect of incoming or de novo–synthesized nucleocapsid on the induction of proinflammatory cytokines and chemokines in macrophages.

(A) Representative cytokine expression induced by SARS-CoV-2 infection in THP-1 macrophage-like cells and CD169- and CD169 + ACE2–overexpressing THP-1 macrophage-like cells. Cells were inoculated with SARS-CoV-2 at MOI 1 and infected for 24 hours. The expression of indicated genes is normalized and quantified by RT-qPCR. The relative fold changes are evaluated by comparisons to uninfected cells and plotted in log₁₀ scale. Data are representative of two independent experiments. Asterisks indicate statistically significant fold change differences of SARS-CoV-2–infected CD169- and CD169 + ACE2–expressing cells as compared to SARS-CoV-2–infected parental cells [two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test: ****P < 0.0001]. (B to F) VLP regulation on TLR7/8 stimulation–induced transcript expression. The indicated cells were incubated with VLPs (Wuhan-Hu-1 strain structural proteins) for 24 hours. They were then stimulated with CL097 for 4 hours. The relative fold changes of the indicated gene are evaluated through RT-qPCR, by comparing to unstimulated cells. Data are representative of two independent experiments. Asterisks indicate statistically significant fold change difference of CL097-treated cells versus VLP + CL097–treated cells (two-way ANOVA and Šidák’s multiple comparisons test: *P < 0.05; ***P < 0.001; ****P < 0.0001). (G) Expression of TNF, CCL4, and CXCL10 transcripts in parental THP-1 macrophage-like cells and cells expressing Wuhan-Hu-1 N protein. The cells were treated with doxycycline (2 μg/ml) for 48 hours to express N. They were then either untreated or stimulated with CL097 for 4 hours. The expression of the indicated genes is normalized and quantified by RT-qPCR. The relative fold changes are evaluated by the comparisons to unstimulated cells and plotted based on 2^−ΔΔCt. Data are representative of two independent experiments (multiple unpaired t tests, using two-stage step-up method of Benjamini, Krieger, and Yekutieli FDR: *P < 0.05; **P < 0.01).