Fig. 5. N-cGAS interaction likely drives hyperinflammation in THP-1 macrophage-like cells.

(A) Schematic of AP-MS used to identify host factors interacting with Delta and Omicron BA.1 variant N proteins in macrophages. This figure was created using BioRender.com with the following credit: Created in BioRender. Li, M. (2026) https://BioRender.com/y4pvy47. (B) High-confidence host factors interacting with SARS-CoV-2 variant N proteins. Thirty-nine proteins passing SAINT BFDR ≤ 0.05 and average spectral count ≥ 11 were analyzed for induction fold changes in Delta N–expressing versus control cells and Omicron BA.1 N-expressing versus control cells. Colors in the PPI represent Delta Log₂ FC versus Omicron BA.1 Log₂ FC (red: stronger interaction with Delta N; blue: stronger interaction with Omicron BA.1 N). Dashed lines indicate interaction with at least one variant N, and solid lines indicate interaction with N proteins from both variants. (C) Functional annotation clustering of high-confidence N interactors. The 39 host proteins were analyzed using DAVID GO and pathway enrichment and grouped by enrichment score. Functional annotation terms are shown on the y axis and FDR on the x axis. Bars exceeding the −log₁₀(FDR) cutoff (red line, FDR = 0.05) indicate significant enrichment. (D). Orthogonal validation of Delta and Omicron BA.1 N protein interactors. HEK293T cells were cotransfected with myc-tagged Delta or Omicron BA.1 N and host factors (3xFLAG-cGAS, 3xFLAG-PABP1, V5-IGF2BP2) or empty vector control. Cells were lysed 24 hours post-transfection and immunoprecipitated with anti–c-myc agarose beads, and binding was detected by immunoblot. (E to G) cGAS inhibition amplifies inflammation-related transcript expression. Cells were pretreated with 10 μM G140 or DMSO for 1 hour before 4-hour CL097 stimulation. Indicated gene expression was measured by RT-qPCR and compared to each cell line’s respective DMSO-treated controls. Data are representative of two independent experiments (two-way ANOVA, multiple comparisons; asterisks indicate statistically significant differences between DMSO and G140 treatments: *P < 0.05; ***P < 0.001; ****P < 0.0001; nonsignificant differences are not shown).